Methods of treating cancer using il-21

ABSTRACT

Methods for treating mammals with cancer using molecules that have an IL-21 functional activity are described. The molecules having IL-21 functional activities include polypeptides that have homology to the human IL-21 polypeptide sequence and proteins fused to a polypeptide with IL-21 functional activity. The molecules can be used as a monotherapy or in combination with other known cancer therapeutics.

REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 10/456,780, filed on Jun. 6, 2003, and is acontinuation-in-part which claims benefit of U.S. ProvisionalApplication Ser. No. 60/387,127, filed on Jun. 7, 2002, and all of whichare incorporated by reference herein.

BACKGROUND OF THE INVENTION

Cytokines generally stimulate proliferation or differentiation of cellsof the hematopoietic lineage or participate in the immune andinflammatory response mechanisms of the body. Examples of cytokineswhich affect hematopoiesis are erythropoietin (EPO), which stimulatesthe development of red blood cells; thrombopoietin (TPO), whichstimulates development of cells of the megakaryocyte lineage; andgranulocyte-colony stimulating factor (G-CSF), which stimulatesdevelopment of neutrophils. These cytokines are useful in restoringnormal blood cell levels in patients suffering from anemia,thrombocytopenia, and neutropenia or receiving chemotherapy for cancer.

The interleukins are a family of cytokines that mediate immunologicalresponses. Central to an immune response is the T cell, which producemany cytokines and adaptive immunity to antigens. Cytokines produced bythe T cell have been classified as type 1 and type 2 (Kelso, A. Immun.Cell Biol. 76:300-317, 1998). Type 1 cytokines include IL-2, IFN-γ,LT-α, and are involved in inflammatory responses, viral immunity,intracellular parasite immunity and allograft rejection. Type 2cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and are involved inhumoral responses, helminth immunity and allergic response. Sharedcytokines between Type 1 and 2 include IL-3, GM-CSF and TNF-α. There issome evidence to suggest that Type 1 and Type 2 producing T cellpopulations preferentially migrate into different types of inflamedtissue.

Mature T cells can activated, i.e., by an antigen or other stimulus, toproduce, for example, cytokines, biochemical signaling molecules, orreceptors that further influence the fate of the T cell population.

B cells can be activated via receptors on their cell surface including Bcell receptor and other accessory molecules to perform accessory cellfunctions, such as production of cytokines.

Natural killer (NK) cells have a common progenitor cell with T cells andB cells, and play a role in immune surveillance. NK cells, whichcomprise up to 15% of blood lymphocytes, do not express antigenreceptors, and therefore do not use MHC recognition as requirement forbinding to a target cell. NK cells are involved in the recognition andkilling of certain tumor cells and virally infected cells. In vivo, NKcells are believed to require activation, however, in vitro, NK cellshave been shown to kill some types of tumor cells without activation.

Lymphomas are malignancies of the lymphatic system, that areheterogenous in etiology, morphology, and clinical course. Lymphomas aregenerally classified as either Hodgkins disease or Non-Hodgkinslymphomas. Hodgkins disease is characterized by giant histocytes,whereas absence of the cells encompasses all non-Hodgkins lymphomas.Lymphocytes, which are the primary component of lymph, can be B celllymphocytes or T cell lymphocytes. Generally, when a lymphoma arisesearly in cell maturation, the malignancy is more aggressive thanmalignancies arising from mature cells. Chemotherapy is usually moreeffective in treating aggressive lymphoma, whereas indolent lymphomascannot be treated as easily and therefore are likely never to be curedas long as the disease remains indolent. For a survey of informationrelating to lymphoma, see, e.g. Lymphoma Treatments and Managing TheirSide Effects, Lymphoma Res. Found. Of Amer., Los Angeles, 2001.

In other aspects, the present invention provides such methods fortreating solid tumors and lymphomas by administrating IL-21 compositionsthat may used as a monotherapy or in combination with chemotherapy,radiation therapy, or other biologics. These and other uses should beapparent to those skilled in the art from the teachings herein.

SUMMARY OF THE INVENTION

Within one aspect, the present invention provides a method of treatingNon-Hodgskins lymphoma comprising administering to a subject in needthereof a therapeutically effective amount of a polypeptide having afunctional activity of IL-21. In certain embodiments, the polypeptidehas been shown to not cause proliferation of isolated cancer cells priorto administration to the subject.

In another aspect, the present invention provides a method of treatingcancer comprising administering to subject a therapeutically effectiveamount of a polypeptide having a functional activity of IL-21, whereinthe cancer is selected from the group of renal cell carcinoma,epithelial carcinoma, breast cancer, prostate cancer, ovarian cancer andcolon cancer. In one embodiment, there is a tumor response. In anotherembodiment, the tumor response is measured as complete response, partialresponse or reduction in time to progression.

In another aspect, the present invention provides a method of treatingNon-Hodgskins lymphoma comprising administering to a subject in needthereof a therapeutically effective amount of a fusion proteincomprising a first polypeptide having a functional activity of IL-21 anda second polypeptide. In other embodiments, the methods provide thecancer is selected from the group of renal cell carcinoma, epithelialcarcinoma, breast cancer, prostate cancer, ovarian cancer and coloncancer. In one embodiment, there is a tumor response. In anotherembodiment, the tumor response is measured as complete response, partialresponse or reduction in time to progression.

DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “cancer” or “cancer cell” is used herein to denote a tissue orcell found in a neoplasm which possesses characteristics whichdifferentiate it from normal tissue or tissue cells. Among suchcharacteristics include but are not limited to: degree of anaplasia,irregularity in shape, indistinctness of cell outline, nuclear size,changes in structure of nucleus or cytoplasm, other phenotypic changes,presence of cellular proteins indicative of a cancerous or pre-cancerousstate, increased number of mitoses, and ability to metastasize. Wordspertaining to “cancer” include carcinoma, sarcoma, tumor, epithelioma,leukemia, lymphoma, polyp, and scirrus, transformation, neoplasm, andthe like.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” denotes apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “level” when referring to immune cells, such as NK cells, Tcells, in particular cytotoxic T cells, B cells and the like, anincreased level is either increased number of cells or enhanced activityof cell function.

The term “level” when referring to viral infections refers to a changein the level of viral infection and includes, but is not limited to, achange in the level of CTLs or NK cells (as described above), a decreasein viral load, an increase antiviral antibody titer, decrease inserological levels of alanine aminotransferase, or improvement asdetermined by histological examination of a target tissue or organ.Determination of whether these changes in level are significantdifferences or changes is well within the skill of one in the art.

The term “neoplastic”, when referring to cells, indicates cellsundergoing new and abnormal proliferation, particularly in a tissuewhere in the proliferation is uncontrolled and progressive, resulting ina neoplasm. The neoplastic cells can be either malignant, i.e. invasiveand metastatic, or benign.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to 10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery thatadministration of IL-21 results in inhibiting proliferation of certainneoplastic cells, either directly or indirectly, thereby limiting thepathological effects caused by specific cancers. In the examples whichfollow, animal models and in vitro assays demonstrate the activity ofIL-21 on biological samples.

A. Description of IL-21 and Its Receptor.

Human IL-21 (SEQ ID NO:1 and SEQ ID NO:2) was designated IL-21, and isdescribed in commonly-owned U.S. Pat. No. 6,307,024, which isincorporated herein by reference. The IL-21 receptor, (previouslydesignated zalpha11) now designated IL-21R (SEQ ID NO:5 and SEQ IDNO:6), and heterodimeric receptor IL-21R/IL-2Rγ are described incommonly-owned WIPO Publication No.s WO 0/17235 and WO 01/77171, whichare incorporated herein by reference. As described in thesepublications, IL-21 was isolated from a cDNA library generated fromactivated human peripheral blood cells (hPBCs), which were selected forCD3. CD3 is a cell surface marker unique to cells of lymphoid origin,particularly T cells.

The amino acid sequence for the IL-21R indicated that the encodedreceptor belonged to the Class I cytokine receptor subfamily thatincludes, but is not limited to, the receptors for IL-2, IL-4, IL-7,IL-15, EPO, TPO, GM-CSF and G-CSF (for a review see, Cosman, “TheHematopoietin Receptor Superfamily” in Cytokine 5(2): 95-106, 1993). Thetissue distribution of the receptor suggests that a target for IL-21 ishematopoietic lineage cells, in particular lymphoid progenitor cells andlymphoid cells. Other known four-helical-bundle cytokines that act onlymphoid cells include IL-2, IL-4, IL-7, and IL-15. For a review offour-helical-bundle cytokines, see, Nicola et al., Advances in ProteinChemistry 52:1-65, 1999 and Kelso, A., Immunol. Cell Biol. 76:300-317,1998.

For IL-21, the secretory signal sequence is comprised of amino acidresidues 1 (Met) to 31 (Gly), and the mature polypeptide is comprised ofamino acid residues 32 (Gln) to 162 (Ser) (as shown in SEQ ID NO: 2). Ingeneral, cytokines are predicted to have a four-alpha helix structure,with helices A, C and D being most important in ligand-receptorinteractions, and are more highly conserved among members of the family.Referring to the human IL-21 amino acid sequence shown in SEQ ID NO:2,an alignment of human IL-21, human IL-15, human IL-4, and human GM-CSFamino acid sequences predicted that IL-21 helix A is defined by aminoacid residues 41-56; helix B by amino acid residues 69-84; helix C byamino acid residues 92-105; and helix D by amino acid residues 135-148;as shown in SEQ ID NO: 2. Structural analysis suggests that the A/B loopis long, the B/C loop is short and the C/D loop is parallel long. Thisloop structure results in an up-up-down-down helical organization. Thecysteine residues are absolutely conserved between IL-21 and IL-15. Thecysteine residues that are conserved between IL-15 and IL-21 correspondto amino acid residues 71, 78, 122 and 125 of SEQ ID NO: 2. Conservationof some of the cysteine residues is also found in IL-2, IL-4, GM-CSF andIL-21 corresponding to amino acid residues 78 and 125 of SEQ ID NO: 2.Consistent cysteine placement is further confirmation of thefour-helical-bundle structure. Also highly conserved in the familycomprising IL-15, IL-2, IL-4, GM-CSF and IL-21 is the Glu-Phe-Leusequence as shown in SEQ ID NO: 2 at residues 136-138. Further analysisof IL-21 based on multiple alignments predicts that amino acid residues44, 47 and 135 (as shown in SEQ ID NO: 2) play an important role inIL-21 binding to its cognate receptor. Moreover, the predicted aminoacid sequence of murine IL-21 (SEQ ID NO:4) shows 57% identity to thepredicted human protein. Based on comparison between sequences of humanand murine IL-21 well-conserved residues were found in the regionspredicted to encode alpha helices A and D.

The corresponding polynucleotides encoding the IL-21 polypeptideregions, domains, motifs, residues and sequences described herein are asshown in SEQ ID NO:1. The amino acid residues comprising helices A, B,C, and D, and loops A/B, B/C and C/D for IL-21 , IL-2, IL-4, IL-15 andGM-CSF are shown in Table 1. TABLE 1 A/B B/C C/D Helix A Loop Helix BLoop Helix C Loop Helix D IL- 41-56 57-68 69-84 85-91 92-105 106-134135-148 SEQ ID 21residues NO: 2 IL-2 36-46 47-52 53-75 76-86 87-99 100-102 103-121 SEQ ID residues NO: 5 IL-4 29-43 44-64 65-83 84-9495-118 119-133 134-151 SEQ ID residues NO: 6 IL-15 45-68 69-83 84-101102-106 107-119  120-133 134-160 SEQ ID residues NO: 7 GM-CSF 30-4445-71 72-81 82-90 91-102 103-119 120-131 SEQ ID residues NO: 8

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human IL-21 and that allelicvariation and alternative splicing are expected to occur. Allelicvariants of this sequence can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures.Allelic variants of the DNA sequence shown in SEQ ID NO:1, includingthose containing silent mutations and those in which mutations result inamino acid sequence changes, are within the scope of the presentinvention, as are proteins which are allelic variants of SEQ ID NO:2.cDNAs generated from alternatively spliced mRNAs, which retain theproperties of the IL-21 polypeptide, are included within the scope ofthe present invention, as are polypeptides encoded by such cDNAs andmRNAs. Allelic variants and splice variants of these sequences can becloned by probing cDNA or genomic libraries from different individualsor tissues according to standard procedures known in the art.

The present invention also provides isolated IL-21 polypeptides thathave a substantially similar sequence identity to the polypeptides ofSEQ ID NO:2, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides comprising atleast 70%, at least 80%, at least 90%, at least 95%, or greater than 95%sequence identity to the sequences shown in SEQ ID NO:2, or theirorthologs. The present invention also includes polypeptides thatcomprise an amino acid sequence having at least 70%, at least 80%, atleast 90%, at least 95% or greater than 95% sequence identity to thesequence of amino acid residues 1 to 162 or 33 to 162 of SEQ ID NO:2.The present invention further includes nucleic acid molecules thatencode such polypeptides. Methods for determining percent identity aredescribed below.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 2 (amino acids are indicated by the standard one-lettercodes).$\frac{{Total}\quad{number}\quad{of}\quad{identical}\quad{matches}}{\begin{bmatrix}{{length}\quad{of}\quad{the}\quad{longer}\quad{sequence}\quad{plus}\quad{the}} \\{{number}\quad{of}\quad{gaps}\quad{introduced}\quad{into}\quad{the}\quad{longer}} \\{{sequence}\quad{in}\quad{order}\quad{to}\quad{align}\quad{the}\quad{two}\quad{sequences}}\end{bmatrix}} \times 100$ TABLE 2 A R N D C Q E G H I L K M F P S T W YV A 4 R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 00 2 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3−1 −3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2−1 −3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3−3 −1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 10 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1−2 −1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2−2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3−3 3 1 −2 1 −1 −2 −2 0 −3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant IL-21. The FASTA algorithm is described by Pearson and Lipman,Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Variant IL-21 polypeptides or polypeptides with substantially similarsequence identity are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table3) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue, a small linker peptide of up toabout 20-25 residues, or an affinity tag. The present invention thusincludes polypeptides of from about 108 to 216 amino acid residues thatcomprise a sequence that is at least 80%, preferably at least 90%, andmore preferably 95%, 96%, 97%, 98%, 99% or more identical to thecorresponding region of SEQ ID NO:2. Polypeptides comprising affinitytags can further comprise a proteolytic cleavage site between the IL-21polypeptide and the affinity tag. Preferred such sites include thrombincleavage sites and factor Xa cleavage sites. TABLE 3 Conservative aminoacid substitutions Basic: arginine lysine histidine Acidic: glutamicacid aspartic acid Polar: glutamine asparagine Hydrophobic: leucineisoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small:glycine alanine serine threonine methionine

Determination of amino acid residues that comprise regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to alignment of multiple sequences withhigh amino acid or nucleotide identity, secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules.

Amino acid sequence changes are made in IL-21 polypeptides so as tominimize disruption of higher order structure essential to biologicalactivity. For example, where the IL-21 polypeptide comprises one or morehelices, changes in amino acid residues will be made so as not todisrupt the helix geometry and other components of the molecule wherechanges in conformation abate some critical function, for example,binding of the molecule to its binding partners, e.g., A and D helices,residues 44, 47 and 135 of SEQ ID NO: 2. The effects of amino acidsequence changes can be predicted by, for example, computer modeling asdisclosed above or determined by analysis of crystal structure (see,e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). Othertechniques that are well known in the art compare folding of a variantprotein to a standard molecule (e.g., the native protein). For example,comparison of the cysteine pattern in a variant and standard moleculescan be made. Mass spectrometry and chemical modification using reductionand alkylation provide methods for determining cysteine residues whichare associated with disulfide bonds or are free of such associations(Bean et al., Anal. Biochem. 201:216-226, 1992; Gray, Protein Sci.2:1732-1748, 1993; and Patterson et al., Anal. Chem. 66:3727-3732,1994). It is generally believed that if a modified molecule does nothave the same cysteine pattern as the standard molecule folding would beaffected. Another well known and accepted method for measuring foldingis circular dichrosism (CD). Measuring and comparing the CD spectragenerated by a modified molecule and standard molecule is routine(Johnson, Proteins 7:205-214, 1990). Crystallography is another wellknown method for analyzing folding and structure. Nuclear magneticresonance (NMR), digestive peptide mapping and epitope mapping are alsoknown methods for analyzing folding and structurally similaritiesbetween proteins and polypeptides (Schaanan et al., Science 257:961-964,1992).

A Hopp/Woods hydrophilicity profile of the IL-21 protein sequence asshown in SEQ ID NO:2 can be generated (Hopp et al., Proc. Natl. Acad.Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquieret al., Protein Engineering 11:153-169, 1998). The profile is based on asliding six-residue window. Buried G, S, and T residues and exposed H,Y, and W residues were ignored. For example, in IL-21, hydrophilicregions include amino acid residues 114-119 of SEQ ID NO: 2, amino acidresidues 101-105 of SEQ ID NO: 2, amino acid residues 126-131 of SEQ IDNO: 2, amino acid residues 113-118 of SEQ ID NO: 2, and amino acidresidues 158-162 of SEQ ID NO: 2.

Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a IL-21 polypeptide, so as not to disruptthe overall structural and biological profile. Of particular interestfor replacement are hydrophobic residues selected from the groupconsisting of Val, Leu and Ile or the group consisting of Met, Gly, Ser,Ala, Tyr and Trp. For example, residues tolerant of substitution couldinclude residues 100 and 103 as shown in SEQ ID NO: 2. Cysteine residuesat positions 71, 78, 122 and 125 of SEQ ID NO: 2, will be relativelyintolerant of substitution.

The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between IL-15, IL-2, IL-4 and GM-CSFwith IL-21. Using methods such as “FASTA” analysis described previously,regions of high similarity are identified within a family of proteinsand used to analyze amino acid sequence for conserved regions. Analternative approach to identifying a variant IL-21 polynucleotide onthe basis of structure is to determine whether a nucleic acid moleculeencoding a potential variant IL-21 gene can hybridize to a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, as discussedabove.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalor biochemical activity as disclosed below to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699 (1996).

The present invention also includes administration of molecules havingthe functional activity of IL-21. Thus, administration of functionalfragments and functional modified polypeptides of IL-21 polypeptides andnucleic acid molecules encoding such functional fragments and modifiedpolypeptides. A “functional” IL-21 or fragment thereof as defined hereinis characterized by its proliferative or differentiating activity, byits ability to induce or inhibit specialized cell functions, inparticular for immune effector cells, such as NK cells, T cells, B cellsand dendritic cells. Functional IL-21 also includes the ability toexhibit anti-cancer and anti-viral effects in vitro or in vivo, or byits ability to bind specifically to an anti-IL-21 antibody or IL-21receptor (either soluble or immobilized). As previously describedherein, IL-21 is characterized by a four-helical-bundle structurecomprising helix A (amino acid residues 41-56), helix B (amino acidresidues 69-84), helix C (amino acid residues 92-105) and helix D (aminoacid residues 135-148), as shown in SEQ ID NO: 2. Thus, the presentinvention further provides fusion proteins encompassing: (a) polypeptidemolecules comprising one or more of the helices described above; and (b)functional fragments comprising one or more of these helices. The otherpolypeptide portion of the fusion protein can contributed by anotherfour-helical-bundle cytokine, such as IL-15, IL-2, IL-4 and GM-CSF, orby a non-native and/or an unrelated secretory signal peptide thatfacilitates secretion of the fusion protein.

Routine deletion analyses of nucleic acid molecules can be performed toobtain functional fragments of a nucleic acid molecule that encodes aIL-21 polypeptide. As an illustration, DNA molecules having thenucleotide sequence of SEQ ID NO:1 or fragments thereof, can be digestedwith Bal31 nuclease to obtain a series of nested deletions. These DNAfragments are then inserted into expression vectors in proper readingframe, and the expressed polypeptides are isolated and tested for IL-21activity, or for the ability to bind anti-IL-21 antibodies or zalpha11receptor. One alternative to exonuclease digestion is to useoligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired IL-21 fragment. Alternatively,particular fragments of a IL-21 gene can be synthesized using thepolymerase chain reaction.

Standard methods for identifying functional domains are well-known tothose of skill in the art. For example, studies on the truncation ateither or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993);Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation 1 Boynton et al.,(eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol.Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meiselet al., Plant Molec. Biol. 30:1 (1996).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204), and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)).

Variants of the disclosed IL-21 nucleotide and polypeptide sequences canalso be generated through DNA shuffling as disclosed by Stemmer, Nature370:389 (1994), Stemmer, Proc. Natl Acad. Sci. USA 91:10747 (1994), andinternational publication No. WO 97/20078. Briefly, variant DNAmolecules are generated by in vitro homologous recombination by randomfragmentation of a parent DNA followed by reassembly using PCR,resulting in randomly introduced point mutations. This technique can bemodified by using a family of parent DNA molecules, such as allelicvariants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-IL-21 antibodies or soluble zalpha11 receptor, canbe recovered from the host cells and rapidly sequenced using modernequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

In addition, the proteins of the present invention (or polypeptidefragments thereof) can be joined to other bioactive molecules,particularly other cytokines, to provide multi-functional molecules. Forexample, one or more helices from IL-21 can be joined to other cytokinesto enhance their biological properties or efficiency of production.

The present invention thus provides a series of novel, hybrid moleculesin which a segment comprising one or more of the helices of IL-21 isfused to another polypeptide. Fusion is preferably done by splicing atthe DNA level to allow expression of chimeric molecules in recombinantproduction systems. The resultant molecules are then assayed for suchproperties as improved solubility, improved stability, prolongedclearance half-life, improved expression and secretion levels, andpharmacodynamics. Such hybrid molecules may further comprise additionalamino acid residues (e.g. a polypeptide linker) between the componentproteins or polypeptides.

Non-naturally occurring amino acids include, without limitation,trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,trans-4-hydroxyproline, N-methylglycine, allo-threonine,methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylicacid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occurring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is typically carried out in acell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993). It can advantageous tostabilize IL-21 to extend the half-life of the molecule, particularlyfor extending metabolic persistence in an active state. To achieveextended half-life, IL-21 molecules can be chemically modified usingmethods described herein. PEGylation is one method commonly used thathas been demonstrated to increase plasma half-life, increasedsolubility, and decreased antigenicity and immunogenicity (Nucci et al.,Advanced Drug Delivery Reviews 6:133-155, 1991 and Lu et al., Int. J.Peptide Protein Res. 43:127-138, 1994).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids can substituted for IL-21 amino acid residues.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a IL-21 polypeptide describedherein. Such fragments or peptides may comprise an “immunogenicepitope,” which is a part of a protein that elicits an antibody responsewhen the entire protein is used as an immunogen. Immunogenicepitope-bearing peptides can be identified using standard methods (see,for example, Geysen et al., Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodiesthat bind with the polypeptides described herein. Hopp/Woodshydrophilicity profiles can be used to determine regions that have themost antigenic potential (Hopp et al., 1981, ibid. and Hopp, 1986,ibid.). In IL-21 these regions include: amino acid residues 114-119,101-105, 126-131, 113-118, and 158-162 of SEQ ID NO: 2.

Antigenic epitope-bearing peptides and polypeptides preferably containat least four to ten amino acids, at least ten to fourteen amino acids,or about fourteen to about thirty amino acids of SEQ ID NO:2 or SEQ IDNO:4. Such epitope-bearing peptides and polypeptides can be produced byfragmenting a IL-21 polypeptide, or by chemical peptide synthesis, asdescribed herein. Moreover, epitopes can be selected by phage display ofrandom peptide libraries (see, for example, Lane and Stephen, Curr.Opin. Immunol. 5:268 (1993); and Cortese et al., Curr. Opin. Biotechnol.7:616 (1996)). Standard methods for identifying epitopes and producingantibodies from small peptides that comprise an epitope are described,for example, by Mole, “Epitope Mapping,” in Methods in MolecularBiology, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc.1992); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages60-84 (Cambridge University Press 1995), and Coligan et al (eds.),Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

Regardless of the particular nucleotide sequence of a variant IL-21polynucleotide, the polynucleotide encodes a polypeptide that ischaracterized by its proliferative or differentiating activity, itsability to induce or inhibit specialized cell functions, or by theability to bind specifically to an anti-IL-21 antibody or zalpha11receptor. More specifically, variant IL-21 polynucleotides will encodepolypeptides which exhibit at least 50% and preferably, greater than70%, 80% or 90%, of the activity of the polypeptide as shown in SEQ IDNO: 2.

For any IL-21 polypeptide, including variants and fusion proteins, oneof ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the genetic code andmethods known in the art.

The present invention further provides a variety of other polypeptidefusions (and related multimeric proteins comprising one or morepolypeptide fusions). For example, a IL-21 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Preferred dimerizing proteins in this regardinclude immunoglobulin constant region domains. Immunoglobulin- IL-21polypeptide fusions can be expressed in genetically engineered cells (toproduce a variety of multimeric IL-21 analogs). Auxiliary domains can befused to IL-21 polypeptides to target them to specific cells, tissues,or macromolecules. For example, a IL-21 polypeptide or protein could betargeted to a predetermined cell type by fusing a IL-21 polypeptide to aligand that specifically binds to a receptor on the surface of thattarget cell. In this way, polypeptides and proteins can be targeted fortherapeutic or diagnostic purposes. A IL-21 polypeptide can be fused totwo or more moieties, such as an affinity tag for purification and atargeting domain. Polypeptide fusions can also comprise one or morecleavage sites, particularly between domains. See, Tuan et al.,Connective Tissue Research 34:1-9, 1996.

Using the methods discussed herein, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptides that havesubstantially similar sequence identity to residues 1-162 or 33-162 ofSEQ ID NO: 2, or functional fragments and fusions thereof, wherein suchpolypeptides or fragments or fusions retain the properties of thewild-type protein such as the ability to stimulate proliferation,differentiation, induce specialized cell function or bind the IL-21receptor or IL-21 antibodies.

The IL-21 polypeptides used in the present invention can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryoticcells, particularly cultured cells of multicellular organisms, arepreferred. Techniques for manipulating cloned DNA molecules andintroducing exogenous DNA into a variety of host cells are disclosed bySambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, andAusubel et al., eds., Current Protocols in Molecular Biology, John Wileyand Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a IL-21 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a IL-21 polypeptide into the secretory pathway of a host cell,a secretory signal sequence (also known as a leader sequence, preprosequence or pre sequence) is provided in the expression vector. Thesecretory signal sequence may be that of IL-21, or may be derived fromanother secreted protein (e.g., t-PA) or synthesized de novo. Thesecretory signal sequence is operably linked to the IL-21 DNA sequence,i.e., the two sequences are joined in the correct reading frame andpositioned to direct the newly synthesized polypeptide into thesecretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain secretory signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-6, 1996).

A wide variety of suitable recombinant host cells includes, but is notlimited to, gram-negative prokaryotic host organisms. Suitable strainsof E. coli include W3110, K12-derived strains MM294, TG-1, JM-107, BL21,and UT5600. Other suitable strains include: BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, ER1647, E. coli K12, E. coli K12 RV308, E. coli K12 C600,E. coli HB101, E. coli K12 C600 R.sub.k-M.sub.k-, E. coli K12 RR1 (see,for example, Brown (ed.), Molecular Biology Labfax (Academic Press1991)). Other gram-negative prokaryotic hosts can include Serratia,Pseudomonas, Caulobacter. Prokaryotic hosts can include gram-positiveorganisms such as Bacillus, for example, B. subtilis and B.thuringienesis, and B. thuringienesis var. israelensis, as well asStreptomyces, for example, S. lividans, S. ambofaciens, S. fradiae, andS. griseofuscus. Suitable strains of Bacillus subtilus include BR151,YB886, MI119, MI120, and B170 (see, for example, Hardy, “BacillusCloning Methods,” in DNA Cloning: A Practical Approach, Glover (ed.)(IRL Press 1985)). Standard techniques for propagating vectors inprokaryotic hosts are well-known to those of skill in the art (see, forexample, Ausubel et al (eds.), Short Protocols in Molecular Biology3^(rd) Edition (John Wiley & Sons 1995); Wu et al, Methods in GeneBiotechnology (CRC Press, Inc. 1997)). In one embodiment, the methods ofthe present invention use IL-21 expressed in the W3110 strain, which hasbeen deposited at the American Type Culture Collection (ATCC) as ATCC#27325.

When large scale production of IL-21 using the expression system of thepresent invention is required, batch fermentation can be used.Generally, batch fermentation comprises that a first stage seed flask isprepared by growing E. coli strains expressing IL-21 in a suitablemedium in shake flask culture to allow for growth to an optical density(OD) of between 5 and 20 at 600 nm. A suitable medium would containnitrogen from a source(s) such as ammonium sulfate, ammonium phosphate,ammonium chloride, yeast extract, hydrolyzed animal proteins, hydrolyzedplant proteins or hydrolyzed caseins. Phosphate will be supplied frompotassium phosphate, ammonium phosphate, phosphoric acid or sodiumphosphate. Other components would be magnesium chloride or magnesiumsulfate, ferrous sulfate or ferrous chloride, and other trace elements.Growth medium can be supplemented with carbohydrates, such as fructose,glucose, galactose, lactose, and glycerol, to improve growth.Alternatively, a fed batch culture is used to generate a high yield ofIL-21 protein. The IL-21 producing E. coli strains are grown underconditions similar to those described for the first stage vessel used toinoculate a batch fermentation.

Following fermentation the cells are harvested by centrifugation,re-suspended in homogenization buffer and homogenized, for example, inan APV-Gaulin homogenizer (Invensys APV, Tonawanda, N.Y.) or other typeof cell disruption equipment, such as bead mills or sonicators.Alternatively, the cells are taken directly from the fermentor andhomogenized in an APV-Gaulin homogenizer. The washed inclusion body prepcan be solubilized using guanidine hydrochloride (5-8 M) or urea (7-8 M)containing a reducing agent such as beta mercaptoethanol (10-100 mM) ordithiothreitol (5-50 mM). The solutions can be prepared in Tris,phopshate, HEPES or other appropriate buffers. Inclusion bodies can alsobe solubilized with urea (2-4 M) containing sodium lauryl sulfate(0.1-2%). In the process for recovering purified IL-21 from transformedE. coli host strains in which the IL-21 is accumulates as refractileinclusion bodies, the cells are disrupted and the inclusion bodies arerecovered by centrifugation. The inclusion bodies are then solubilizedand denatured in 6 M guanidine hydrochloride containing a reducingagent. The reduced IL-21 is then oxidized in a controlled renaturationstep. Refolded IL-21 can be passed through a filter for clarificationand removal of insoluble protein. The solution is then passed through afilter for clarification and removal of insoluble protein. After theIL-21 protein is refolded and concentrated, the refolded IL-21 proteinis captured in dilute buffer on a cation exchange column and purifiedusing hydrophobic interaction chromatography.

It is preferred to purify the polypeptides of the present invention to≧80% purity, more preferably to ≧90% purity, even more preferably ≧95%purity, and particularly preferred is a pharmaceutically pure state,that is greater than 99.9% pure with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. Preferably, a purified polypeptideis substantially free of other polypeptides, particularly otherpolypeptides of animal origin.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which bind to IL-21 proteins or polypeptides.Exemplary assays are described in detail in Antibodies: A LaboratoryManual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press,1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant IL-21protein or polypeptide.

The methods of the present invention also contemplate using chemicallymodified IL-21 compositions, in which a IL-21 polypeptide is linked witha polymer. Illustrative IL-21 polypeptides are soluble polypeptides thatlack a functional transmembrane domain, such as a mature IL-21polypeptide. Typically, the polymer is water soluble so that the IL-21conjugate does not precipitate in an aqueous environment, such as aphysiological environment. An example of a suitable polymer is one thathas been modified to have a single reactive group, such as an activeester for acylation, or an aldehyde for alkylation, In this way, thedegree of polymerization can be controlled. An example of a reactivealdehyde is polyethylene glycol propionaldehyde, or mono-(C1-C10)alkoxy, or aryloxy derivatives thereof (see, for example, Harris, et al,U.S. Pat. No. 5,252,714). The polymer may be branched or unbranched.Moreover, a mixture of polymers can be used to produce IL-21 conjugates.

IL-21 conjugates used for therapy can comprise pharmaceuticallyacceptable water-soluble polymer moieties. Suitable water-solublepolymers include polyethylene glycol (PEG), monomethoxy-PEG,mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG,tresyl monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonatePEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxideco-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, dextran, cellulose, or other carbohydrate-based polymers.Suitable PEG may have a molecular weight from about 600 to about 60,000,including, for example, 5,000, 12,000, 20,000 and 25,000. A IL-21conjugate can also comprise a mixture of such water-soluble polymers.

B. The Use of IL-21 for Treating Cancer

Differentiation is a progressive and dynamic process, beginning withpluripotent stem cells and ending with terminally differentiated cells.Pluripotent stem cells that can regenerate without commitment to alineage express a set of differentiation markers that are lost whencommitment to a cell lineage is made. Progenitor cells express a set ofdifferentiation markers that may or may not continue to be expressed asthe cells progress down the cell lineage pathway toward maturation.Differentiation markers that are expressed exclusively by mature cellsare usually functional properties such as cell products, enzymes toproduce cell products, and receptors. The stage of a cell population'sdifferentiation is monitored by identification of markers present in thecell population.

There is evidence to suggest that factors that stimulate specific celltypes down a pathway towards terminal differentiation ordedifferentiation affect the entire cell population originating from acommon precursor or stem cell. Thus, the present invention includesstimulating or inhibiting the proliferation of lymphoid cells,hematopoietic cells and epithelial cells.

IL-21 was isolated from tissue known to have important immunologicalfunction and which contain cells that play a role in the immune system.IL-21 is expressed in CD3+ selected, activated peripheral blood cells,and it has been shown that IL-21 expression increases after T cellactivation. Moreover, results of experiments described in the Examplessection herein demonstrate that polypeptides of the present inventionhave an effect on the growth/expansion and/or differentiated state of NKcells or NK progenitors. Factors that both stimulate proliferation ofhematopoietic progenitors and activate mature cells are generally known.NK cells are responsive to IL-2 alone, but proliferation and activationgenerally require additional growth factors. For example, it has beenshown that IL-7 and Steel Factor (c-kit ligand) were required for colonyformation of NK progenitors. IL-15+IL-2 in combination with IL-7 andSteel Factor was more effective (Mrózek et al., Blood 87:2632-2640,1996). However, unidentified cytokines may be necessary forproliferation of specific subsets of NK cells and/or NK progenitors(Robertson et. al., Blood 76:2451-2438, 1990). A composition comprisingIL-21 and IL-15 stimulates NK progenitors and NK cells, with evidencethat this composition is more potent than previously described factorsand combinations of factors. Moreover, IL-21 promotes NK-cell expansion,and IL-21 can largely overcome the inhibitory effects of IL-4 on NK-cellgrowth, it synergizes with IL-2 to promote NK cell growth, and IL-21selectively promotes the expression of IFN-γ and depresses IL-13expression. These data suggest that IL-21 have an indirect role intreating solid tumors, metastatic tumors and lymphomas by stimulatingthe immune effector cells resulting in anti-lymphoma activity. Inaddition, for certain cancerous cells where the IL-21 receptor isexpressed, the anticancer effect of IL-21 can be direct.

Additional evidence demonstrates that IL-21 affects proliferation and/ordifferentiation of T cells and B cells in vivo. It is shown that IL-21can either inhibit or enhance the proliferation of normal B cellsdepending on the nature of the co-stimulus provided the cells. IL-21inhibits the proliferation of some B cell lines, but not others eventhough most non-responder cell lines express IL-2IR as measured byspecific IL-21 binding. Many human B cell lines will grow in and killSCID mice Bonnefoix et al., Leukemia and Lymphoma 25:169-178, 1997).Examples herein describe three B-cell lines which are inhibited by IL-21and three B cell lines which did not respond to IL-21. All of the celllines were IL-21R positive, and were put into SCID mice to determine ifIL-21 could prolong the survival of lymphoma bearing animals. IL-21exhibited significant efficacy against the three cell lines whoseproliferation was inhibited in vitro. In a separate experiment, NK-celldepletion of the SCID mice failed to abrogate the IL-21 effect in theIM-9 model, suggesting that NK-cells are not required for the efficacyof IL-21 in this model.

Assays measuring differentiation include, for example, measuring cellmarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference). Alternatively, IL-21 polypeptide itself can serveas an additional cell-surface or secreted marker associated withstage-specific expression of a tissue. As such, direct measurement ofIL-21 polypeptide or its receptors expressed on cancer cells, or itsloss of expression in a tissue as it differentiates, can serve as amarker for differentiation of tissues.

The classification of lymphomas most commonly used is the REALclassification system (Ottensmeier, Chemico-Biological Interactions135-136:653-664, 2001.) Specific immunological markers have beenidentified for classifications of lymphomas. For example, follicularlymphoma markers include CD20+, CD3−, CD10+, CD5−; Small lymphocyticlymphoma markers include CD20+, CD3−, CD10−, CD5+, CD23+; marginal zoneB cell lymphoma markers include CD20+, CD3−, CD10−, CD23−; diffuse largeB cell lymphoma markers include CD20+, CD3−; mantle cell lymphomamarkers include CD20+, CD3−, CD10−, CD5+, CD23+; peripheral T celllymphoma markers include CD20−, CD3+; primary mediastinal large B celllymphoma markers include CD20+, CD3−, lymphoblastic lymphoma markersinclude CD20−, CD3+, Tdt+, and Burkitt's lymphoma markers include CD20+,CD3−, CD10+, CD5− (Decision Resourses, Non-Hodgkins Lymphoma, Waltham,Mass., February 2002).

Primary lymphoma specimens are routinely acquired by biopsy of nodal orextra nodal tumors in the diagnosis of lymphoma. For some lymphoidneoplasms, in particular Chronic Lymphocytic leukemia (CLL), themalignant cells can be acquired from the patient's blood. One method fortesting whether a specific lymphoma or patient is amenable to treatmentwith IL-21 is culturing lymphoma cells. Biopsy or blood specimens can beprepared for tissue culture by a combination of methods well known tothose skilled in the art. For example, the samples can be prepared bymincing, teasing, enzymatic digestion or density gradient centrifugation(ficoll) (Jacob et al., Blood 75(5):1154-1162, 1990). The tumor cellsare then labeled with a fluorescent DNA stain such as carboxyfluoresceindiacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, Oreg.) andcultured in IL-21. The distribution of tumor cells that have undergone 1or more rounds of cell division can be quatitated by flow cytometry,with the cells losing ½ of their CFSE intensity with each round ofreplication. The proportion of inviable cells and the number ofapoptotic cells are also analysed analyzed for the effect of IL-21 using7-AAD and annexin-V staining. The number of surviving cells, thedistribution of CFSE staining and the per cent of cells that areapoptotic can all be used to determine whether IL-21 promotes orinhibits the growth and survival of a given malignant specimen. In suchan analysis the lymphoma cells are distinguished from normal cellscontaminating the specimen by a combination of a B-cell lineage specificmarker, immunoglobulin light chain lambda and kappa specific antibodyand light scatter properties. For CLL specimens CD5 staining can also beutilized as an aid in defining the malignant cells. As the proportion ofcells proliferating in vitro is likely to be very low, it is essentialthat the tumor cells be distinguished from normal cells. A data analysismethod such as flow cytometry that can measure multiple parameters onindividual cells is preferred.

One exemplary method for determination specific lymphoma sensitivity toIL-21 uses biopsy or blood cells cultured in serum free medium or inmedium containing serum or plasma, preferably fetal bovine serum orhuman serum, at varying doses of IL-21, generally in a range from 0.1 to10 nM, and including a negative control. At various time points, forexample 1 ,2, 4 and 7 days cells are harvested and subjected to flowcytometric methods to determine the distribution of cells that havedivided 1 or more times (CFSE intensity), the proportion of inviablecells(7-AAD staining; Hausner et al., J. Immunol. Methods 247(1-2):175-186, 2001) and the number of apoptotic cells (annexin-Vstaining; Lagneaux et al., Br. J. Hematol. 112 (2):344-352, 2001). Thenumber of surviving cells, the distribution of CFSE staining and the percent of cells that are apoptotic can all be used to determine whetherIL-21 promotes or inhibits the growth and survival of a given malignantspecimen. In such an analysis the lymphoma cells are distinguished fromnormal cells contaminating the specimen by a combination of a B-celllineage specific marker, immunoglobulin light chain lambda and kappaspecific antibody and light scatter properties. For CLL specimens CD5staining can also be utilized as an aid in defining the malignant cells.As the proportion of cells proliferating in vitro may be very low, it iscritical that the tumor cells be distinguished from normal cells in thedata analysis and is why a method such as flow cytometry that canmeasure multiple parameters on individual cells is useful.

Such testing of individual tumor specimens will provide basis to choosewhich patients are likely to respond to IL-21 favorably and for whichpatients IL-21 may be contraindicated. Patients whose malignantlymphocytes proliferate more slowly in response to IL-21 than in controlcultures or die more rapidly than control cultures would be consideredas candidates for IL-21 therapy. Similarly, a patient whose malignantcells proliferation rate or survival is enhanced by IL-21 in vitrowould, in general, not be candidates for IL-21 therapy (except as notedbelow). Once data are accumulated that demonstrates a strong correlationbetween a particular type of lymphoma (for example, follicular lymphomaor CLL) and sensitivity to IL-21 in vitro, the need to test all patientswithin such a subgroup for their response to IL-21 may be obviated,provided that no patients are found within the group that exhibitincreased proliferation in response to IL-21 in vitro.

Similarly, direct measurement of IL-21 polypeptide, or its loss ofexpression in a tissue can be determined in a tissue or in cells as theyundergo tumor progression. Increases in invasiveness and motility ofcells, or the gain or loss of expression of IL-21 in a pre-cancerous orcancerous condition, in comparison to normal tissue, can serve as adiagnostic for transformation, invasion and metastasis in tumorprogression. As such, knowledge of a tumor's stage of progression ormetastasis will aid the physician in choosing the most proper therapy,or aggressiveness of treatment, for a given individual cancer patient.Methods of measuring gain and loss of expression (of either mRNA orprotein) are well known in the art and described herein and can beapplied to IL-21 expression. For example, appearance or disappearance ofpolypeptides that regulate cell motility can be used to aid diagnosisand prognosis of prostate cancer (Banyard, J. and Zetter, B. R., Cancerand Metast. Rev. 17:449-458, 1999). As an effector of cell motility,IL-21 gain or loss of expression may serve as a diagnostic for lymphoidcancers.

As discussed above, the IM-9 mouse model for cancer demonstrated thatantitumor activity is not NK cell dependent. There are several syngeneicmouse models that have been developed to study the influence ofpolypeptides, compounds or other treatments on tumor progression. Inthese models, tumor cells passaged in culture are implanted into mice ofthe same strain as the tumor donor. The cells will develop into tumorshaving similar characteristics in the recipient mice, and metastasiswill also occur in some of the models. Appropriate tumor models for ourstudies include the Lewis lung carcinoma (ATCC No. CRL-1642) and B16melanoma (ATCC No. CRL-6323), amongst others. These are both commonlyused tumor lines, syngeneic to the C57BL6/J mouse, that are readilycultured and manipulated in vitro. Tumors resulting from implantation ofeither of these cell lines are capable of metastasis to the lung inC57BL6/J mice. The Lewis lung carcinoma model has recently been used inmice to identify an inhibitor of angiogenesis (O'Reilly M S, et al. Cell79: 315-328,1994). C57BL6/J mice are treated with an experimental agenteither through daily injection of recombinant protein, agonist orantagonist or a one time injection of recombinant adenovirus. Three daysfollowing this treatment, 10⁵ to 10⁶ cells are implanted under thedorsal skin. Alternatively, the cells themselves can be infected withrecombinant adenovirus, such as one expressing IL-21, beforeimplantation so that the protein is synthesized at the tumor site orintracellularly, rather than systemically. The mice normally developvisible tumors within 5 days. The tumors are allowed to grow for aperiod of up to 3 weeks, during which time they may reach a size of1500-1800 mm³ in the control treated group. Tumor size and body weightare carefully monitored throughout the experiment. At the time ofsacrifice, the tumor is removed and weighed along with the lungs and theliver. The lung weight has been shown to correlate well with metastatictumor burden. As an additional measure, lung surface metastases arecounted. The resected tumor, lungs and liver are prepared forhistopathological examination, immunohistochemistry, and in situhybridization, using methods known in the art and described herein. Theinfluence of the expressed polypeptide in question, e.g., IL-21, on theability of the tumor to recruit vasculature and undergo metastasis canthus be assessed. In addition, aside from using adenovirus, theimplanted cells can be transiently transfected with IL-21. Use of stableIL-21 transfectants as well as use of induceable promoters to activateIL-21 expression in vivo are known in the art and can be used in thissystem to assess IL-21 induction of metastasis. Moreover, purified IL-21or IL-21 conditioned media can be directly injected in to this mousemodel, and hence be used in this system. For general reference see,O'Reilly M S, et al. Cell 79:315-328, 1994; and Rusciano D, et al.Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.

The activity of IL-21 and its derivatives (conjugates) on growth anddissemination of tumor cells derived from human hematologic malignanciescan be measured in vivo. Several mouse models have been developed inwhich human tumor cells are implanted into immunodeficient mice(collectively referred to as xenograft models); see, for example, CattanA R, Douglas E, Leuk. Res. 18:513-22, 1994 and Flavell, D J,Hematological Oncology 14:67-82, 1996. The characteristics of thedisease model vary with the type and quantity of cells delivered to themouse, and several disease models are known in the art. In an example ofthis model, tumor cells (e.g. Raji cells (ATCC No. CCL-86)) would bepassaged in culture and about 1×10⁶ cells injected intravenously intosevere combined immune deficient (SCID) mice. Such tumor cellsproliferate rapidly within the animal and can be found circulating inthe blood and populating numerous organ systems. Therapies designed tokill or reduce the growth of tumor cells using IL-21 or its derivatives,agonists, conjugates or variants can be tested by administration ofIL-21 compounds to mice bearing the tumor cells. Efficacy of treatmentis measured and statistically evaluated as increased survival within thetreated population over time. Tumor burden may also be monitored overtime using well-known methods such as flow cytometry (or PCR) toquantitate the number of tumor cells present in a sample of peripheralblood. For example, therapeutic strategies appropriate for testing insuch a model include direct treatment with IL-21 or related conjugatesor antibody-induced toxicity based on the interaction of IL-21 with itsreceptor(s), or for cell-based therapies utilizing IL-21 or itsderivatives, agonists, conjugates or variants. The latter method,commonly referred to as adoptive immunotherapy, would involve treatmentof the animal with components of the human immune system (i.e.lymphocytes, NK cells, bone marrow) and may include ex vivo incubationof cells with IL-21 with or without other immunomodulatory agentsdescribed herein or known in the art.

The activity of IL-21 on immune (effector) cell-mediated tumor celldestruction can be measured in vivo, using the murine form of the IL-21protein (SEQ ID NO:2) in syngeneic mouse tumor models. Several suchmodels have been developed in order to study the influence ofpolypeptides, compounds or other treatments on the growth of tumor cellsand interaction with their natural host, and can serve as models fortherapeutics in human disease. In these models, tumor cells passaged inculture or in mice are implanted into mice of the same strain as thetumor donor. The cells will develop into tumors having similarcharacteristics in the recipient mice. For reference, see, for example,van Elsas et al., J. Exp. Med. 190:355-66, 1999; Shrikant et al.,Immunity 11:483-93, 1999; and Shrikant et al., J. Immunol. 162:2858-66,1999. Appropriate tumor models for studying the activity of IL-21 onimmune (effector) cell-mediated tumor cell destruction include theB16-F10 melanoma (ATCC No. CRL-6457), and the EG.7 thymoma (ATCC No.CRL-2113), described herein, amongst others. These are both commonlyused tumor cell lines, syngeneic to the C57BL6 mouse, which are readilycultured and manipulated in vitro.

In an example of an in vivo model, the tumor cells (e.g. B16-F10melanoma (ATCC No. CRL-6475) are passaged in culture and about 100,000cells injected intravenously into C57BL6 mice. In this mode ofadministration, B16-F10 cells will selectively colonize the lungs. Smalltumor foci are established and will grow within the lungs of the hostmouse. Therapies designed to kill or reduce the growth of tumor cellsusing IL-21 or its derivatives, agonists, conjugates or variants can betested by administration of compounds to mice bearing the tumor cells.Efficacy of treatment is measured and statistically evaluated byquantitation of tumor burden in the treated population at a discretetime point, two to three weeks following injection of tumor cells.Therapeutic strategies appropriate for testing in such a model includedirect treatment with IL-21 or its derivatives, agonists, conjugates orvariants, or cell-based therapies utilizing IL-21 or its derivatives,agonists, conjugates or variants. The latter method, commonly referredto as adoptive immunotherapy, would involve treatment of the animal withimmune system components (i.e. lymphocytes, NK cells, dendritic cells orbone marrow, and the like) and may include ex vivo incubation of cellswith IL-21 with or without other immunomodulatory agents describedherein or known in the art.

Another syngeneic mouse tumor cell line can used to test the anti-cancerefficacy of IL-21 and to identify the immune (effector) cell populationresponsible for mediating this effect. EG.7ova is a thymoma cell linethat has been modified (transfected) to express ovalbumin, an antigenforeign to the host. Mice bearing a transgenic T cell receptor specificfor EG.7ova are available (OT-1 transgenics, Jackson Laboratory). CD8 Tcells isolated from these animals (OT-1 T cells) have been demonstratedto kill EG.7 cells in vitro and to promote rejection of the tumor invivo. EG.7ova cells can be passaged in culture and about 1,000,000 cellsinjected intraperitoneal into C57BL6 mice. Multiple tumor sites areestablished and grow within the peritoneal cavity. Therapies designed tokill or reduce the growth of tumor cells using IL-21 or its derivatives,agonists, conjugates or variants can be tested by administration ofcompounds to mice bearing the tumor cells. OT-1 T cells can beadministered to the mice to determine if their activity is enhanced inthe presence of IL-21. Efficacy of treatment is measured andstatistically evaluated by time of survival in the treated populations.Therapeutic strategies appropriate for testing in such models includedirect treatment with IL-21 or its derivatives, agonists, conjugates orvariants, or cell-based therapies utilizing IL-21 or its derivatives,agonists, conjugates or variants. Ex vivo treatment of cytotoxicT-lymphocytes (CTL) could also be used to test the IL-21 in thecell-based strategy.

Analysis of IL-21 efficacy for treating certain specific types ofcancers are preferably made using animals that have been shown tocorrelate to other mammalian disease, particularly human disease. AfterIL-21 is administered in these models evaluation of the effects on thecancerous cells or tumors is made. Xenografts are used for mostpreclinical work, using immunodeficient mice. For example, a syngeneicmouse model for ovarian carcinoma utilizes a C57BL6 murine ovariancarcinoma cell line stably overexpressing VEGF16 isoform and enhancedgreen fluorescent protein (Zhang et al., Am. J. Pathol. 161:2295-2309,2002). Renal cell carcinoma mouse models using Renca cell injectionshave been shown to establish renal cell metastatic tumors that areresponsive to treatment with immunotherapeutics such as IL-12 and IL-2(Wigginton et al., J. of Nat. Cancer Inst. 88:38-43, 1996). A colorectalcarcinoma mouse model has been established by implanting mouse colontumor MC-26 cells into the splenic subcapsule of BALB/c mice (Yao etal., Cancer Res. 63 (3):586-586-592, 2003). Animmunotherapeutic-responsive mouse model for breast cancer has beendeveloped using a mouse that spontaneously develops tumors in themammary gland and demonstrates peripheral and central tolerance to MUCI(Mukherjee et al., J. Immunotherapy 26:47-42, 2003). To test theefficacy of IL-21 in prostate cancer, animal models that closely mimichuman disease have been developed. A transgenic adenocarcinoma of themouse prostate model (TRAMP) is the most commonly used syngeneic model(Kaplan-Lefko et al., Prostate 55 (3):219-237, 2003; Kwon et al., PNAS96:15074-15079, 1999; Arap et al., PNAS 99:1527-1531, 2002).

IL-21 will be useful in treating tumorgenesis, enhancing CTLs and NKactivity, and therefore are useful in the treatment of cancer. Inaddition to direct and indirect effects on CTLs and NK cells, as shownin several tumor models described herein, IL-21 inhibits IL-4 stimulatedproliferation of anti-IgM stimulated normal B-cells and a similar effectis observed in B-cell tumor lines suggesting that there can betherapeutic benefit in treating patients with the IL-21 in order toinduce the B cell tumor cells into a less proliferative state.

The ligand could be administered in combination with other agentsalready in use including both conventional chemotherapeutic agents aswell as immune modulators such as interferon alpha. Alpha/betainterferons have been shown to be effective in treating some leukemiasand animal disease models, and the growth inhibitory effects of INF-αand IL-21 are additive for at least one B-cell tumor-derived cell line.Establishing the optimal dose level and scheduling for IL-21 is done bya combination of means, including the pharmacokinetics andpharmacodynamics of IL-21, the sensitivity of human B-cell lines andprimary lymphoma specimens to IL-21 in vitro, effective doses in animalmodels and the toxicity of IL-21. Optimally, to have a direct anti-tumoreffect the concentration of IL-21 in plasma should reach a level that invitro is maximally active against B-cell lymphoma cell lines and primarylymphomas. In addition the optimal and minimum times of exposure toIL-21 to elicit a growth inhibitory or apoptotic responses can bemodeled in vitro with cell lines and primary tumor cells. Directpharmacokinetic measurements done in primates and clinical trials canthen be used to predict theoretical doses in patients that achieveplasma IL-21 levels that are of sufficient magnitude and duration toachieve a biological response in patients. In addition IL-21 stimulatesa variety of responses in normal lymphocytes, such that surrogatemarkers can be employed to measure the biological activty of IL-21 oneffector cells in patients.

Since lymphoma patients are treated with a variety of chemotherapeuticdrugs and drug combinations, developing a protocol to integrate IL-21into an existing standard treatment regimen may result in improvedtherapeutic outcome. The effect of combining chemotherapy drugs andIL-21 is primarily modeled with IL-21 sensitive human B-cell lines invitro, measuring cell proliferation, cell viability, and apoptosis. Timeand dose dependent response curves to chemotherapeutic drugs (e.g.chlorambucil, etoposide, or fludaribine) are established for individualcell lines. IL-21 is then tested over a wide range of concentrationsunder suboptimal conditions of each chemotherapy drug. The order ofexposure of the cells to IL-21 versus a chemotherapy agent maysignificantly affect the outcome of the interaction with the cell linetested. As such, the IL-21 should be introduced to the cultures inseveral manners to find the optimal mode of treatment. This shouldinclude, for example, prior treatment with IL-21 for several hours toseveral days (0, 4, 24, 48 and 72 hours ), followed by a wash out ofIL-21 and the addition of a sub-optimal dose/exposure time of achemotherapy drug. After 1-3 days, analysis of the culture for cellviability, proliferation and apoptosis is made. In a variation to theabove experiment, the IL-21 is not washed out prior to the addition ofthe chemotherpy drug. The complete set of conditions to test would alsoinclude the simultaneous treatment of cells with IL-21 and achemotherapy drug with varying time of IL-21 wash out, as well asdelayed (from a few hours to several days) addition of IL-21 until afterexposure to a chemotherapy drug. The timing and concentration of IL-21exposure that gives a maximal reduction in target celloutgrowth/viability or maximum increase in apoptotic response will thenbe considered optimal for further testing in animal models or the designof a clinical protocol. The combination of IL-21 with chemotherapeuticdrugs in vitro using cell lines whose growth is stimulated by IL-21,such as RPMI-1788, could be done to identify drugs that eliminate thepotential adverse affects of IL-21 that might be encountered in a subsetof patients. Such drugs would be identified from those known to haveactivity against lymphomas and selected on the basis of their ability invitro to prevent enhanced growth and or survival of RPMI 1788 orsimilarly IL-21 responsive cell lines. In this way IL-21 therapy, whencombined with selected chemotherapeutic regimens would be of benefit tothose patients whose malignancy is sensitve to IL-21 mediated growthsuppression, while protecting any patients that might otherwise respondunfavorably to IL-21 monotherapy.

Lymphoma patients are also treated with biologics, such as RITUXAN™,IL-2 and interferon. Those biologic agents that have a direct inhibitoryeffect on the tumor and are not largely dependent on effector cells fortheir activity can be modeled in a manner similar to the in vitroexperiments above for their interaction with IL-21. For example,RITUXAN™ binds to lymphoma cells and can induce apoptosis directly invitro, but is also capable of inducing a variety of effector mechanismssuch as complement dependent cytotoxity and antibody dependentcell-mediated cytotoxicity (ADCC). Therefore, it is possible to defineconditions in vitro in which IL-21 and RITUXAN™ interact in synergy toinhibit lymphoma growth or stimulate apoptosis. The use of a xenogeneichuman lymphoma model in SCID mice has the potential to measure a broaderrange of potential interactions that involve host effector mechanismsbetween RITUXAN™ (or some other biologic agent) and IL-21. To determineif there is significant synergy between IL-21 and another anti-tumorbiologic in a xenogeneic lymphoma SCID mouse model, IL-21 and the otherbiologic are tested under conditions that yield marginal therapeuticresults with either agent alone. [100] IL-21 and IL-2 exhibit synergy intheir effects on NK-cells in vitro with respect to IFN-γ production andproliferation. In addition, high dose IL-2 therapy is highly toxic andrequires extensive hospitalization. Many low dose regimens of IL-2 havebeen tested, and found to be well tolerated, but with little evidence ofanti-tumor efficacy (Atkins, Semin. Oncol. 29 (3 Suppl. 7):12, 2002).The combination of low dose IL-2 with IL-21 therefore may be clinicallyuseful by augmenting the immune system stimulation of low dose IL-2while providing a direct anti-lymphoma effect. The effects of combiningIL-2 and IL-21 are studied in mouse syngeneic lymphoma models or in SCIDmouse xenogeneic human lymphoma models as described herein. The relativeeffects on effector cell activation and toxicity of combining IL-2 andIL-21 at different doses can be determined in normal primates tooptimize a dosing level and schedule to avoid the need to hospitalizepatients.

For those patients whose malignant lymphocytes are stimulated toproliferate in vitro in response to IL-21, a course of IL-21 could becontraindicated (in the absence of combination with other drugs asdiscussed above) unless the malignant cells have a very low turn overrate in vivo, such as CLL. The relatively quiescent state of CLL cellsmay be related to the resistance of this disease to chemotherapy. Insuch cases, patients could be treated by pulsing them with IL-21 justprior to the administration of chemotherapeutic drug(s). The optimaltiming of dosing of IL-21 and chemotherapy could be modeled in vitro topredict how long after exposure to IL-21 the malignant cells becomemaximally sensitive to specific chemotherapeutic drugs.

The present invention provides a method of reducing proliferation of aneoplastic B or T cells comprising administering to a mammal with a B orT cell lymphoma an amount of a composition of IL-21 sufficient to reduceproliferation of the B or T lymphoma cells. In other embodiments, thecomposition can comprise at least one other cytokine selected from thegroup consisting of IL-2, IL-15, IL-4, IL-18, GM-CSF, Flt3 ligand,interferon, or stem cell factor.

In another aspect, the present invention provides a method of reducingproliferation of a neoplastic B or T cells comprising administering to amammal with a B or T cell neoplasm an amount of a composition of IL-21antagonist sufficient to reducing proliferation of the neoplastic B or Tcells. In other embodiments, the composition can comprise at least oneother cytokine selected from the group consisting of IL-2, IL-15, IL-4,IL-18, GM-CSF, Flt3 ligand, interferon, or stem cell factor.Furthermore, the IL-21 antagonist can be a ligand/toxin fusion protein.

A IL-21-saporin fusion toxin, or other IL-21-toxin fusion, can beemployed against a similar set of leukemias and lymphomas, extending therange of leukemias that can be treated with IL-21. Moreover, suchIL-21-toxin fusions can be employed against other cancers wherein IL-21binds its receptors. Fusion toxin mediated activation of the IL-21receptor provides two independent means to inhibit the growth of thetarget cells, the first being identical to the effects seen by theligand alone, and the second due to delivery of the toxin throughreceptor internalization. The lymphoid restricted expression pattern ofthe IL-21 receptor suggests that the ligand-saporin conjugate can betolerated by patients.

When treatment for malignancies includes allogeneic bone marrow or stemcell transplantion, IL-21 can be valuable in enhancement of thegraft-vs-tumor effect. IL-21 stimulates the generation of lytic NK cellsfrom marrow progenitors and stimulates the proliferation of T-cellsfollowing activation of the antigen receptors. Therefore, when patientsreceive allogenic marrow transplants, IL-21 will enhance the generationof anti-cancer responses, with or without the infusion of donorlymphocytes.

Modern methods for cancer immunotherapy are based on the principle thatthe immune system can detect and defend against spontaneous tumors.Evidence supporting the concept of “Immunological surveillance” (see,Burnet F M Lancet 1: 1171-4, 1967), comes in part from epidemiologicalstudies indicating that the incidence of cancer increases in patientsthat are immunocomprised by disease, such as infection (see, Klein G.Harvey Lect. 69:71-102, 1975; and Kuper et al., J. Intern. Med.248:171-83, 2000), or following medical interventions such as bonemarrow ablation (see, Birkeland et al., Lancet 355:1886-7, 2000; andPenn I, Cancer Detect Prev. 18:241-52, 1994). Experiments performed ingene-targeted mice also show that the immune system modulatessusceptibility to spontaneous tumors in aged mice (see, Smyth, M. etal., J. Exp. Med. 192:755-760, 2000; and Davidson, W. et al., J. Exp.Med. 187: 1825-1838, 1998) or following exposure to chemical carcinogens(see, Peng, S et al., J. Exp. Med. 184: 1149-1154, 1996; Kaplan, D. etal., Proc. Nat. Acad Sci. USA 95:7556-7561, 1998; and Shankaran V. etal., Nature 410:1107-1111, 2001). Proof that immune recognition oftumors occurs frequently in tumor bearing hosts comes from theidentification of T-cells that are reactive to a broad range of tumorassociated antigens including differentiation antigens, mutationalantigens, tissue-specific antigens, cancer-testis antigens, selfantigens that are over expressed in tumors, and viral antigens (Boon T.et al., Immunol. Today 18:267-8, 1997.). In addition, B-cells are knownto produce high titers of circulating IgG antibodies that recognizethese same classes of tumor antigens (Stockert E. et al., J. Exp. Med.187:1349-54, 1998;. Sahin U et al.,. Curr. Opin. Immunol. 9:709-16,1997; and Jäger, E. et al., Proc. Nat. Acad. Sci. USA 97:12198-12203,2000), and NK cells have been isolated that can recognize and kill tumorcells that express various stress-related genes (Bauer, S et al.,Science 285:727-729, 1999).

The concept that immunotherapy can be an effective method for treatingcancer is firmly established in experimental animal models, and whilethe methodologies are much less advanced for human subjects, there is astrong suggestion that the immune system can be stimulated to rejectestablished disease. The very first attempt at cancer immunotherapy wasreported by William Coley in 1893 who, using extracts of pyrogenicbacteria, achieved anti-cancer responses most likely through theinduction of systemic inflammation and cell-mediated immunity (Coley WB. The treatment of malignant tumors by repeated inoculations oferysipelas. With a report of ten original cases. 1893, Clin Orthop.262:3-11, 1991). In more modern times five generalized strategies havebeen employed to increase the numbers of effector cells and/or modulatetheir anti-cancer activity (reviewed in Rosenberg, S A. (Ed.),Principles and practice of the biologic therapy of cancer., 3^(rd)edition, Lippincott Williams & Wilkins, Philadelphia, Pa., 2000):cytokine therapy, cell transfer therapy, monoclonal antibody therapy,cancer vaccines, and gene therapy. To date, each method has showneffectiveness in mediating an anti-cancer response although thedurability of these responses, with a few exceptions, is mostlytemporary. This fact reflects our limited understanding of tumorimmunology and argues that improvements in the technology await theutilization of previously unrecognized elements of the anti-cancerresponse. The present invention provides such an element to improve ourunderstanding of tumor immunology as well as provide polypeptides thatare therapeutically useful in treating and preventing human cancers.

One requirement for achieving sustained immunity and durable clinicalresponses is the amplification in the level, i.e., in the numbers andactivity of the cells that mediate tumor killing. Thus, new factors thatmediate their effects on lymphocytes including cytotoxic T-cells (CTLs),NK cells, and B-cells, as well myeloid cells such as neutrophils andmonocytic cells will improve anti-cancer activity. IL-21 is a product ofactivated CD4⁺ “helper” T-cells which are required for both humoral andcell-mediated immunity and for sustaining long-term memory to antigenicre-challenge (U.S. Pat. No. 6,307,024; Parrish-Novak J et al., Nature408:57-63, 2000). The receptor for IL-21 is expressed on cells thatmediate anti-cancer responses and previous experiments have shown thatIL-21 can stimulate the proliferation of these cell types in vitro(commonly-owned WIPO Publication No.s WO 0/17235 and WO 01/77171).Additional experiments affirm these IL-21 activities in vivo.

IL-21 polypeptides for the methods of the present invention are shown tostimulate CTL and NK cells against tumors in vivo in animal modelsresulting in decreased tumor burden and tumor cells, and increasedsurvival. IL-21 can hence be used in therapeutic anti-cancerapplications in humans. As such, IL-21 anti-cancer activity is useful inthe treatment and prevention of human cancers. Such indications includebut are not limited to the following: Carcinomas (epithelial tissues),Sarcomas of the soft tissues and bone (mesodermal tissues), Adenomas(glandular tissues), cancers of all organ systems, such as liver(hepatoma) and kidney (renal cell carcinomas), CNS (gliomas,neuroblastoma), and hematological cancers, viral associated cancers(e.g., associated with retroviral infections, HPV, hepatitis B and C,and the like), lung cancers, endocrine cancers, gastrointestinal cancers(e.g., biliary tract cancer, liver cancer, pancreatic cancer, stomachcancer and colorectal cancer), genitourinary cancers (e.g., prostatecancer bladder cancer, renal cell carcinoma), gynecologic cancers (e.g.,uterine cancer, cervical cancer, ovarian cancer) breast, and othercancers of the reproductive system, head and neck cancers, and others.Of particular interest are hematopoietic cancers, including but notlimited to, lymphocytic leukemia, myeloid leukemia, Hodgkin's lymphoma,Non-Hodgkins lymphomas, chronic lymphocytic leukemia, and otherleukemias and lymphomas. Moreover IL-21 can be used therapeutically incancers of various non-metastatic as wells as metastatic stages such as“Stage 1” Localized (confined to the organ of origin); “Stage 2”Regional; “Stage 3” Extensive; and “Stage 4” Widely disseminatedcancers. In addition, IL-21 can be used in various applications forcancer, immunotherapy, and in conjunction with chemotherapy and thelike.

Administration of IL-21 using the methods of the present invention willresult in a tumor response. While each protocol may define tumorresponse accessments differently, exemplary guidelines can be found inClinical Research Associates Manual, Southwest Oncology Group, CRAB,Seattle, Wash., Oct. 6, 1998, updated August 1999. According to the CRAManual (see, chapter 7 “Response Accessment”), tumor response means areduction or elimination of all measurable lesions or metastases.Disease is generally considered measurable if it comprisesbidimensionally measurable lesions with clearly defined margins bymedical photograph or X-ray, computerized axial tomography (CT),magnetic resonance imaging (MRI), or palpation. Evaluable disease meansthe disease comprises unidimensionally measurable lesions, masses withmargins not clearly defined, lesion with both diameters less than 0.5cm, lesions on scan with either diameter smaller than the distancebetween cuts, palpable lesions with diameter less than 2 cm, or bonedisease. Non-evaluable disease includes pleural effusions, ascites, anddisease documented by indirect evidence. Previously radiated lesionswhich have not progressed are also generally considered non-evaluable.

The criteria for objective status are required for protocols to accesssolid tumor response. A representative criteria includes the following:(1) Complete Response (CR) defined as complete disappearance of allmeasurable and evaluable disease. No new lesions. No disease relatedsymptoms. No evidence of non-evaluable disease; (2) Partial Response(PR) defined as greater than or equal to 50% decrease from baseline inthe sum of products of perpendicular diameters of all measureablelesions. No progression of evaluable disease. No new lesions. Applies topatients with at least one measurable lesion; (3) Progression defined as50% or an increase of 10 cm² in the sum of products of measurablelesions over the smallest sum observed using same techniques asbaseline, or clear worsening of any evaluable disease, or reappearanceof any lesion which had disappeared, or appearance of any new lesion, orfailure to return for evaluation due to death or deteriorating condition(unless unrelated to this cancer); (4) Stable or No Response defined asnot qualifying for CR, PR, or Progression. (See, Clinical ResearchAssociates Manual, supra.)

Examples of methods for using IL-21 in the treatment of cancer include,but are not limited to, the following:

1) IL-21 can be used as a single agent for direct inhibitory activityagainst tumors that express the IL-21 receptor (U.S. Pat. No. 6,307,024;WIPO Publication No.s WO 0/17235 and WO 01/77171). Such activity isshown herein. Administration in a pharmaceutical vehicle for therapeuticuse can be achieved using methods in the art and described herein.

2) IL-21 can be conjugated to a toxic compound that binds and killstumor cells that express IL-21 receptor such as B-cell lymphomas, T-celllymphomas and NK cell lymphomas. The toxic compound can be a smallmolecule drug like calichaemicin used in a manner similar to theanti-CD33 antibody+drug conjugate, MYLOTARG™, that is used to treatacute myelogeous leukemia (for example, See, Sievers E L et al.,. J ClinOncol. 19:3244-54, 2001; and Bernstein I D Clin. Lymphoma Suppl1:S9-S11, 2002); or a radioisotope like ¹²⁵I (Kaminski M S, et al.. J.Clin. Oncol. 19:3918-28, 2001) or ⁹⁰Y (Reviewed in Gordon L I et al.,Semin. Oncol. (1 Suppl 2):87-92, 2002) that has been attached to ananti-CD20 antibody used for the treatment of Non-Hogkin's lymophoma; ora naturally occurring protein toxin such as Ricin A (Lynch T J Jr, etal., J. Clin. Oncol. 15:723-34, 1997) or diphtheria B toxin that wasmade as a fusion protein with IL-2 for the treatment of cutaneous T-celllymphoma (Talpur R et al., Leuk. Lymphoma 43:121-6, 2002). Theattachment of these toxic compounds to IL-21 might occur throughchemical conjugation (Rapley R. Mol. Biotechnol. 3:139-54, 1995) orgenetic recombination (Foss F M. Clin. Lymphoma Suppl 1:S27-31, 2000).Such toxin conjugates with IL-21, for example IL-21-saporin conjugates,are shown to kill various tumors in vivo and in vitro (U.S. Pat. No.6,307,024; and described herein).

3) IL-21 can be used as an immunostimulatory agent for cancermonotherapy. A variety of cytokines such as IL-2, IL-4, IL-6, IL-12,IL-15, and interferon, are known to stimulate anti-cancer responses inanimal models via stimulation of the immune system (reviewed inRosenberg, S A ibid.). Moreover IL-21 is shown to also stimulate theimmune system (U.S. Pat. No. 6,307,024; and described herein). Cytokinemonotherapy is an accepted practice for human cancer patients. Forexample, the use of IL-2 and IFN-α are used for the treatments ofmetastatic melanoma and renal cell carcinoma (e.g., see, Atkins M B etal., J. Clin. Oncol. 17:2105-16, 1999; Fyfe G et al., J. Clin. Oncol.13:688-96, 1995; and Jonasch E, and Haluska F G, Oncologist 6:34-55,2001). The mechanism of action of these cytokines includes, but is notlimited to, an enhancement of a Th1 cell-mediated responses includingdirect tumor cell killing by CD8+ T-cells and NK cells. IL-21 is shownto similarly enhance Th1 cell-mediated responses including direct tumorcell killing by CTLs, e.g., CD8+ T-cells, and NK cells in vivo and invitro as described herein. Thus, IL-21 of the present invention can beused therapeutically or clinically to actively kill tumor cells in humandisease, and to regulate these activities, as well as in additionalanti-cancer responses.

4) IL-21 can be used as an immunostimulatory agent in combination withchemotherapy, radiation, and myeloablation. In addition to working aloneto boost anti-cancer immunity in patients, IL-21 can work in synergywith standard types of chemotherapy or radiation. For instance, inpreclinical models of lymphoma and renal cell carcinoma, the combinationof IL-2 with doxorubicin (Ehrke M J et al., Cancer Immunol. Immunother.42:221-30, 1996), or the combinations of IL-2 (Younes E et al., CellImmunol. 165:243-51, 1995) or IFN-α (Nishisaka N et al., Cytokines CellMol Ther. 6:199-206, 2000) with radiation provided superior results overthe use of single agents. In this setting, IL-21 can further reducetumor burden and allow more efficient killing by the chemotherapeutic.Additionally, lethal doses of chemotherapy or radiation followed by bonemarrow transplantation or stem cell reconstitution could reduce tumorburden to a sufficiently small level (ie. minimal residual disease) tobetter allow an IL-21 mediated anti-cancer effect. Examples of this typeof treatment regimen include the uses of IL-2 and IFN-α to modifyanti-cancer responses following myeloablation and transplantation(Porrata L F et al., Bone Marrow Transplant. 28:673-80, 2001; Slavin S,and Nagler A. Cancer J. Sci. Am. Suppl 1:S59-67, 1997; and Fefer A etal., Cancer J. Sci. Am. Suppl 1:S48-53, 1997). In the case of lymphomaand other cancers, depending on when IL-21 is used relative to thechemotherapeutic agents, IL-21 may be employed to directly synergizewith the chemotherapeutic agent's effect on the tumor cells oralternatively employed after the chemotherapy to stimulate the immunesystem. Those skilled in the art would design a protocol to takeadvantage of both possibilities.

5) IL-21 can be used as a tissue protective agent in combination withstandard forms of chemotherapy or methods that ablate bone marrow. IL-21regulates the proliferation and differentiation of cells. As a result,IL-21 can protect various tissues and organs from the toxicitiesassociated with commonly used chemotherapies and radiation. As anexample, gut epithelium expresses IL-15 receptor and experiments inanimal models show that IL-15 protects intestinal epithelium fromchemotherapy induced toxicity and prevents morbidity (Shinohara H etal., Clin. Cancer Res. 5:2148-56, 1999; Cao S et al., Cancer Res.58:3270-4, 1998; and Cao S et al., Cancer Res. 58: 1695-9, 1998). Inaddition to protecting against damage, the proliferative effects ofIL-21 can accelerate tissue regeneration following drug-inducedtoxicity. Relevant examples of this type of activity include theenhanced reconstitution of the immune system stimulated by IL-7following bone marrow transplantation (Alpdogan O et al., Blood98:2256-65, 2001; and Mackall C L et al., Blood 97:1491-7, 2001) and theuse of G-CSF to treat neutropenia following chemotherapy (Lord, B I etal., Clin. Cancer Res. 7:2085-90, 2001; and Holmes F A et al., J. Clin.Oncol. 20:727-31, 2002). Because IL-21 is shown to enhance proliferationand differentiation of hematopoietic and lymphoid cells, IL-21 of thepresent invention can be used therapeutically or clinically to aid inrecovery as well as enhance the chemotherapeutic dosage schemes uponadministration of chemotherapeutic agents in human disease.

6) IL-21 can be used in combination with other immunomodulatorycompounds including various cytokines and co-stimulatory/inhibitorymolecules. The immunostimulatory activity of IL-21 in mediating ananti-cancer response can be enhanced in patients when IL-21 is used withother classes of immunomodulatory molecules. These could include, butare not limited to, the use of additional cytokines. For instance, thecombined use of IL-2 and IL-12 shows beneficial effects in T-celllymphoma, squamous cell carcinoma, and lung cancer (Zaki M H et al., J.Invest. Dermatol. 118:366-71, 2002; Li D et al., Arch. Otolaryngol. HeadNeck Surg. 127:1319-24, 2001; and Hiraki A et al., Lung Cancer35:329-33, 2002). In addition IL-21 could be combined with reagents thatco-stimulate various cell surface molecules found on immune-basedeffector cells, such as the activation of CD137 (Wilcox R A et al., J.Clin. Invest. 109:651-9, 2002) or inhibition of CTLA4 (Chambers C A etal., Ann. Rev. Immunol. 19:565-94, 2001). Alternatively, IL-21 could beused with reagents that induce tumor cell apoptosis by interacting withTRAIL-related receptors (Takeda K et al., J. Exp. Med. 195:161-9, 2002;and Srivastava R K, Neoplasia 3:535-46, 2001). Such reagents includeTRAIL ligand, TRAIL ligand-Ig fusions, anti-TRAIL antibodies, and thelike.

7) IL-21 can be used in combination with Monoclonal Antibody Therapy.Treatment of cancer with monoclonal antibodies is becoming a standardpractice for many tumors including Non-Hodgkins lymphoma (RITUXAN™),forms of leukemia (MYLOTARG™), breast cell carcinoma (HERCEPTIN™), andcolon carcinoma (ERBITUX™). One mechanism by which antibodies mediate ananti-cancer effect is through a process referred to asantibody-dependent cell-mediated cytotoxicity (ADCC) in whichimmune-based cells including NK cells, macrophages and neutrophils killthose cells that are bound by the antibody complex. Due to itsimmunomodulatory activity, IL-21 can be used to enhance theeffectiveness of antibody therapy. Examples of this type of treatmentparadigm include the combination use of RITUXAN™ and either IL-2, IL-12,or IFN-α for the treatment of Hodgkin's and Non-Hodgkin's lymphoma(Keilholz U et al., Leuk. Lymphoma 35:641-2,. 1999; Ansell S M et al.,Blood 99:67-74, 2002; Carson W E et al., Eur. J. Immunol. 31:3016-25,2001; and Sacchi S et al., Haematologica 86:951-8., 2001). Similarly,Because IL-21 is shown to enhance proliferation and differentiation ofhematopoietic and lymphoid cells, as well as NK cells, IL-21 of thepresent invention can be used therapeutically or clinically to enhancethe enhance the activity and effectiveness of antibody therapy in humandisease.

8) IL-21 can be used in combination with cell adoptive therapy. Onemethod used to treat cancer is to isolate anti-cancer effector cellsdirectly from patients, expand these in culture to very high numbers,and then to reintroduce these cells back into patients. The growth ofthese effector cells, which include NK cells, LAK cells, andtumor-specific T-cells, requires cytokines such as IL-2 (Dudley M E etal., J. Immunother. 24:363-73, 2001). Given its growth stimulatoryproperties on lymphocytes, IL-21 could also be used to propagate thesecells in culture for subsequent re-introduction into patients in need ofsuch cells. Following the transfer of cells back into patients, methodsare employed to maintain their viability by treating patients withcytokines such as IL-2 (Bear H D et al., Cancer Immunol. Immunother.50:269-74, 2001; and Schultze J L et al., Br. J. Haematol. 113:455-60,2001). Again, IL-21 can be used following adoptive therapy to increaseeffector cell function and survival.

9) IL-21 can be used in combination with tumor vaccines. The majorobjective of cancer vaccination is to elicit an active immune responseagainst antigens expressed by the tumor. Numerous methods for immunizingpatients with cancer antigens have been employed, and a variety oftechniques are being used to amplify the strength of the immune responsefollowing antigen delivery (reviewed in Rosenberg, S A ibid). Methods inwhich IL-21 can be used in combination with a tumor vaccine include, butare not limited to, the delivery of autologous and allogeneic tumorcells that either express the IL-21 gene or in which IL-21 is deliveredin the context of a adjuvant protein. Similarly, IL-21 can be deliveredin combination with injection of purified tumor antigen protein, tumorantigen expressed from injected DNA, or tumor antigen peptides that arepresented to effector cells using dendritic cell-based therapies.Examples of these types of therapies include the use of cytokines likeIL-2 in the context of vaccination with modified tumor cells (Antonia SJ et al., J. Urol. 167:1995-2000, 2002; and Schrayer D P et al., Clin.Exp. Metastasis 19:43-53, 2002), DNA (Niethammer A G et al., Cancer Res.61:6178-84,2001), and dendritic cells (Shimizu K et al., Proc. Nat.Acad. Sci U S A 96:2268-73, 1999). Similarly, IL-21 can be used as ananti-cancer vaccine adjuvant.

10) IL-21 can be used in the context of gene therapy. Gene therapy canbe broadly defined as the transfer of genetic material into a cell totransiently or permanently alter the cellular phenotype. Numerousmethods are being developed for delivery of cytokines, tumor antigens,and additional co-stimulatory molecules via gene therapy to specificlocations within tumor patients (reviewed in Rosenberg, S A ibid). Thesemethodologies could be adapted to use IL-21 DNA or RNA, or IL-21 couldbe used as a protein adjuvant to enhance immunity in combination with agene therapy approach as described herein.

The tissue distribution of a receptor for a given cytokine offers astrong indication of the potential sites of action of that cytokine.Northern analysis of IL-21 receptor revealed transcripts in humanspleen, thymus, lymph node, bone marrow, and peripheral bloodleukocytes. Specific cell types were identified as expressing IL-21receptors, and strong signals were seen in a mixed lymphocyte reaction(MLR) and in the Burkitt's lymphoma Raji. The two monocytic cell lines,THP-1 (Tsuchiya et al., Int. J. Cancer 26:171-176, 1980) and U937(Sundstrom et al., Int. J. Cancer 17:565-577, 1976), were negative.

IL-21 receptor is expressed at relatively high levels in the MLR, inwhich peripheral blood mononuclear cells (PBMNC) from two individualsare mixed, resulting in mutual activation. Detection of high levels oftranscript in the MLR but not in resting T or B cell populationssuggests that IL-21 receptor expression may be induced in one or morecell types during activation. Activation of isolated populations of Tand B cells can be artificially achieved by stimulating cells with PMAand ionomycin. When sorted cells were subjected to these activationconditions, levels of IL-21 receptor transcript increased in both celltypes, supporting a role for this receptor and IL-21 in immuneresponses, especially in autocrine and paracrine T and B cell expansionsduring activation. IL-21 may also play a role in the expansion of moreprimitive progenitors involved in lymphopoiesis.

IL-21 receptor was found to be present at low levels in resting T and Bcells, and was upregulated during activation in both cell types.Interestingly, the B cells also down-regulate the message more quicklythan do T cells, suggesting that amplitude of signal and timing ofquenching of signal are important for the appropriate regulation of Bcell responses.

IL-21 in concert with IL-15 expands NK cells from bone marrowprogenitors and augments NK cell effector function. IL-21 alsoco-stimulates mature B cells stimulated with anti-CD40 antibodies, butinhibits B cell proliferation to signals through IgM. IL-21 enhances Tcell proliferation in concert with a signal through the T cell receptor,and overexpression in transgenic mice leads to lymphopenia and anexpansion of monocytes and granulocytes, as described herein.

IL-21 polypeptides and proteins can also be used ex vivo, such as inautologous marrow culture. Briefly, bone marrow is removed from apatient prior to chemotherapy or organ transplant and treated withIL-21, optionally in combination with one or more other cytokines. Thetreated marrow is then returned to the patient after chemotherapy tospeed the recovery of the marrow or after transplant to suppress graftvs. Host disease. In addition, the proteins of the present invention canalso be used for the ex vivo expansion of marrow or peripheral bloodprogenitor (PBPC) cells. Prior to treatment, marrow can be stimulatedwith stem cell factor (SCF) to release early progenitor cells intoperipheral circulation. These progenitors can be collected andconcentrated from peripheral blood and then treated in culture withIL-21, optionally in combination with one or more other cytokines,including but not limited to SCF, IL-2, IL-4, IL-7, IL-15, IL-18, orinterferon, to differentiate and proliferate into high-density lymphoidcultures, which can then be returned to the patient followingchemotherapy or transplantation.

The present invention provides a method for expansion of hematopoieticcells and hematopoietic cell progenitors comprising culturing bonemarrow or peripheral blood cells with a composition comprising an amountof IL-21 sufficient to produce an increase in the number of lymphoidcells in the bone marrow or peripheral blood cells as compared to bonemarrow or peripheral blood cells cultured in the absence of IL-21. Inother embodiments, the hematopoietic cells and hematopoietic progenitorcells are lymphoid cells. In another embodiment, the lymphoid cells areNK cells or cytotoxic T cells. Furthermore, the composition can alsocomprise at least one other cytokine selected from the group consistingof IL-2, IL-15, IL-4, GM-CSF, Flt3 ligand and stem cell factor.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1

Mouse IL-21 is Active in Mouse Bone Marrow Assay

A. Isolation of Non-adherent Low Density Marrow Cells:

Fresh mouse femur aspirate (marrow) was obtained from 6-10 week old maleBalb/C or C57BL/6 mice. The marrow was then washed with RPMI+10% FBS(JRH, Lenexa Kans.; Hyclone, Logan Utah) and suspended in RPMI+10% FBSas a whole marrow cell suspension. The whole marrow cell suspension wasthen subjected to a density gradient (Nycoprep, 1.077, Animal; GibcoBRL) to enrich for low density, mostly mononuclear, cells as follows:The whole marrow cell suspension (About 8 ml) was carefully pipetted ontop of about 5 ml Nycoprep gradient solution in a 15 ml conical tube,and then centrifuged at 600×g for 20 minutes. The interface layer,containing the low density mononuclear cells, was then removed, washedwith excess RPMI+10% FBS, and pelleted by centrifugation at 400×g for5-10 minutes. This pellet was resuspended in RPMI+10% FBS and plated ina T-75 flask at approximately 10⁶ cells/ml, and incubated at 37° C. 5%CO₂ for approximately 2 hours. The resulting cells in suspension wereNon-Adherent Low Density (NA LD) Marrow Cells.

B. 96-Well Assay

NA LD Mouse Marrow Cells were plated at 25,000 to 45,000 cells/well in96 well tissue culture plates in RPMI+10% FBS+1 ng/mL mouse Stem CellFactor (mSCF) (R&D Systems, Minneapolis, Minn.), plus 5% conditionedmedium from one of the following: (1) BHK 570 cells expressing mouseIL-21 (U.S. Pat. No. 6,307,024), (2) BHK 570 cells expressing humanIL-21 (U.S. Pat. No. 6,307,024), or (3) control BHK 570 cells containingvector and not expressing either Ligand. These cells were then subjectedto a variety of cytokine treatments to test for expansion ordifferentiation of hematopoietic cells from the marrow. To test, theplated NA LD mouse marrow cells were subjected to human Interleukin-15(hIL-15) (R&D Systems), or one of a panel of other cytokines (R&DSystems). Serial dilution of hIl-15, or the other cytokines, weretested, with 2-fold serial dilution from about 50 ng/ml down to about6025 ng/ml concentration. After 8 to 12 days the 96-well assays werescored for cell proliferation by Alamar blue assay as described in U.S.Pat. No. 6,307,024.

C. Results from the 96-well NA LD Mouse Marrow Assay

Conditioned media from the BHK cells expressing both mouse and humanIL-21 acted in synergy with hIL-15 to promote the expansion of apopulation of hematopoietic cells in the NA LD mouse marrow. Thisexpansion of hematopoietic cells was not shown with control BHKconditioned medium plus IL-15. The population hematopoietic cellsexpanded by the mouse IL-21 with hIL-15, and those hematopoietic cellsexpanded by the human IL-21 with hIL-15, were further propagated in cellculture. These hematopoietic cells were stained with a Phycoerythrinlabeled anti-Pan NK cell antibody (Pharmingen) and subjected to flowcytometry analysis, which demonstrated that the expanded cells stainedpositively for this natural killer (NK) cell marker.

The same 96-well assay was run, using fresh human marrow cells boughtfrom Poietic Technologies, Gaithersburg, Md. Again, in conjunction withIL-15, the mouse and human IL-21 expanded a hematopoietic cellpopulation that stained positively for the NK cell marker using theantibody disclosed above.

Example 2

IL-21 Transgenic Mice

A. Generation of Transgenic Mice Expressing Human and Mouse IL-21

DNA fragments from transgenic vectors (U.S. Pat. No. 6,307,024)containing 5′ and 3′ flanking sequences of the respective promoter (MT-1liver-specific promoter (mouse IL-21 (U.S. Pat. No. 6,307,024) orlymphoid specific LCK promoter (mouse and human IL-21 (U.S. Pat. No.6,307,024), the rat insulin II intron, IL-21 cDNA and the human growthhormone poly A sequence were prepared and used for microinjection intofertilized B6C3fl (Taconic, Germantown, N.Y.) murine oocytes, using astandard microinjection protocol. See, Hogan, B. et al., Manipulatingthe Mouse Embryo. A Laboratory Manual Cold Spring Harbor LaboratoryPress, 1994.

Eight transgenic mice expressing human IL-21 from the lymphoid-specificEμLCK promoter were identified among 44 pups. Four of these were pupsthat died and 4 grew to adulthood. Expression levels were fairly low inthese animals. Twenty transgenic mice expressing mouse IL-21 from thelymphoid-specific EμLCK promoter were identified among 77 pups. All 20grew to adulthood. Expression levels were fairly low in these animals.Three transgenic mice expressing mouse IL-21 from the liver-specificMT-1 promoter were identified among 60 pups. Two of these pups died and1 grew to adulthood. Expression levels were fairly low in these animals.Tissues were prepared and histologically examined as describe below.

B. Microscopic Evaluation of Tissues from Transgenic Mice

Spleen, thymus, and mesenteric lymph nodes were collected and preparedfor histologic examination from transgenic animals expressing human andmouse IL-21 (Example 2A). Other tissues which were routinely harvestedincluded the following: Liver, heart, lung, kidney, skin, mammary gland,pancreas, stomach, small and large intestine, brain, salivary gland,trachea, espohogus, adrenal, pituitary, reproductive tract, accessorymale sex glands, skeletal muscle including peripheral nerve, and femurwith bone marrow. The tissues were harvested from a neonatal pup whichdied unexpectedly, and several adult transgenic mice, as describedbelow. Samples were fixed in 10% buffered formalin, routinely processed,embedded in paraffin, sectioned at 5 microns, and stained withhematoxylin and eosin. The slides were examined and scored as toseverity of tissue changes (0=none, 1=mild, 2=moderate, 3=severe) by aboard certified veterinary pathologist blinded to treatment.

The pup and 2 female adult mice expressing the human IL-21, and 3 of the6 male adult mice expressing the mouse IL-21 showed inflammatoryinfiltrates in many of the tissues examined. The organs affected variedsomewhat from mouse to mouse. The inflammatory infiltrate was composedprimarily of neutrophils and macrophages in varying numbers andproportions and was generally mild to moderate degree in severity.Moreover, these animals showed changes in lymphoid organs, includingmoderate to severe lymphopenia in the spleen and thymus (human and mouseIL-21 transgenics); and severe lymphopenia (human IL-21 transgenics), ormild to severe suppurative to pyogranulomatous lymphadenitis (mouseIL-21 transgenics) in lymph nodes. In addition, increased extramedullaryhematopoiesis was evident in the spleens. These changes were notobserved in age-matched control mice.

C. Flow Cytometric Analysis of Tissues from Transgenic Mice overExpressing IL-21

Transgenic animals over expressing either human or mouse zalpha11ligand(Example 2A) were sacrificed for flow cytometric analysis of peripheralblood, thymus, lymph node, bone marrow, and spleen.

Cell suspensions were made from spleen, thymus and lymph nodes byteasing the organ apart with forceps in ice cold culture media (500 mlRPMI 1640 Medium (JRH Biosciences. Lenexa, Kans.); 5 ml 100× L-glutamine(Gibco BRL. Grand Island, N.Y.); 5 ml 100× Na Pyruvate (Gibco BRL); 5 ml100× Penicillin, Streptomycin, Neomycin (PSN) (Gibco BRL) and thengently pressing the cells through a cell strainer (Falcon, VWR Seattle,Wash.). Peripheral blood (200 ml) was collected in heparinized tubes anddiluted to 10 ml with HBSS containing 10 U Heparin/ml. Erythrocytes wereremoved from spleen and peripheral blood preparations by hypotoniclysis. Bone marrow cell suspensions were made by flushing marrow fromfemurs with ice cold culture media. Cells were counted and tested forviability using Trypan Blue (GIBCO BRL, Gaithersburg, Md.). Cells wereresuspended in ice cold staining media (HBSS, 1% fetal bovine serum,0.1% sodium azide) at a concentration of ten million per milliliter.Blocking of Fc receptor and non-specific binding of antibodies to thecells was achieved by adding 10% normal goat sera and Fc Block(Pharmingen, La Jolla, Calif.) to the cell suspension.

Cell suspensions were mixed with equal volumes of fluorochrome labeledmonoclonal antibodies (PharMingen), incubated on ice for 60 minutes andthen washed twice with ice cold wash buffer (PBS, 1% fetal bovine serum,0.1% sodium azide) prior to resuspending in 400 ml wash buffercontaining 1 mg/ml 7-AAD (Molecular Probes, Eugene, Oreg.) as aviability marker in some samples. Flow data was acquired on aFACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose,Calif.). Both acquisition and analysis were performed using CellQuestsoftware (BD Immunocytometry Systems).

The transgenic animals that expressed either the human or mouse IL-21 atthe highest levels had dramatically altered cell populations in alllymphoid organs analyzed. Changes seen included complete loss of thymiccellularity, complete absence of CD45R positive B cells and increasedsize and cellularity of spleens. Both spleen and bone marrow hadincreased numbers of myeloid sized cells, which was accounted for byincreases in both monocytes and neutrophils. The pan NK cell marker(DX5) was increased in many populations. Moderate expressing foundershad less dramatic but still significant changes consistent with thephenotype seen in the high expressers. Mice with the lowest level ofexpression had neither a significant increase in myeloid cells nordecrease in B cells numbers. They did show significant changes inthymocyte populations with decreases in CD4+CD8+ double positive cellsand increases in both CD4 and CD8 single positive cells.

Example 3

IL-21 Purified Recombinant Human Protein

Dose-Response Study in Normal Mice

A. Summary

Normal six week old female C57B1/6 (Harlan Sprague Dawley, Indianapolis,Ind.). mice were treated by intraperitoneal injection once daily foreither four or eight days with one of four dose levels of purifiedrecombinant human IL-21 (U.S. Pat. No. 6,307,024) at 0.1, 0.5, 5 or 50μg/mouse/day or with vehicle as a control. Body weights and bodytemperatures were monitored daily. On either day four or day nine, fourof the eight mice from each protein treatment group and five of the tenmice in the vehicle control group were sacrificed. Blood, bone marrowand tissues were harvested and analyzed. Potential perturbations inlymphoid tissues were examined, as well as general physiologic andtoxicological parameters.

There was no evidence of toxicity of human IL-21 protein at any of thedoses tested. Body weights and temperatures were unchanged. There wereno apparent changes in clinical chemistry parameters. However, therewere consistent findings relating to increased percentages of myeloidlineage cells in bone marrow, spleen and peripheral blood in micetreated with the highest dose of IL-21 compared to the vehicle control.There was a statistically significant increase in myeloid lineage sizedcells identified by flow cytometric analysis of spleen homogenate in thehigh-dose group. The spleens of the two highest dose groups werestatistically significantly larger than the other groups. Onhistopathologic examination, however, only a marginal increase inextramedullary hematopoiesis was seen in the highest dose group. Therewas a statistically significant increase in the myeloid to erythroidratio of the bone marrow in the highest dose group compared to the othergroups. Finally, there were increases seen in peripheral blood both intotal white blood cell counts and in the percentage of monocytes in thesame group.

B. Dosing Solution Preparation

Purified recombinant human IL-21 (U.S. Pat. No. 6,307,024) was dilutedinto sterile phosphate buffered saline (GibcoBRL, Grand Island, N.Y.) atconcentrations to deliver 50, 5, 0.5 or 0. 1 micrograms of protein in0.1 ml of PBS vehicle. The doses for the first four days were made onday 0 and frozen in a frosty −20° C. freezer prior to use. The doses fordays five through eight were made on day five and frozen as above.Aliquots of the same PBS were similarly frozen for the vehicle treatedcontrol group. On the day of administration the appropriate aliquotswere thawed and 0.1 ml of solution was injected intraperitoneally intothe mice each day for either four or eight days.

Study Design

The mice were six weeks old at the start of the study. Each treatmentgroup consisted of eight mice, except for the vehicle control group thatincluded ten mice. One half of the mice in each treatment group weresacrificed after four days of treatment and the other half after eightdays.

Before treatment each day, each mouse was weighed and her bodytemperature recorded using the Portable Programmable Notebook System(BMDS, Inc, Maywood, N.J.), by scanning the mouse for identificationnumber and body temperature from transponders implanted subcutaneously(IPTT-100, BMDS, Maywood, N.J.).

At sacrifice, tissues harvested to assess white blood cell populationsby flow cytometric analysis included bone marrow, thymus and spleen.FACS analysis of the lymphoid organs and bone marrow was performed withthe FACSCalibur, (Becton Dickinson, Mansfield, Mass.). The tissuesharvested for histologic examination for signs of toxicity of theprotein included: spleen, thymus, liver, kidney, adrenal gland, heartand lungs. All tissues fixed for histology were kept at 4° C. overnightin 10% Normal Buffered Saline (NBF) (Surgipath, Richmond, Ill.). Thefollowing day the NBF was replaced with 70% ethanol and the tissuesreturned to 4° C. until processing for histology.

The tissues were processed and stained for Hematoxylin and Eosin inhouse, then sent to a contract pathologist for histopathologic analysis.Blood was collected for complete blood cell counts (CBC) and serumchemistry profiles. The CBC's were analyzed in-house with the Cell Dyn3500 Hematology Analyzer (Abbott Diagnostics Division, Abbott Park,Ill.) and manual differential white blood cell counts were analyzed atPhoenix Central Laboratory, (Everett, Wash.). The serum was kept frozenat −20° C. until submission to Phoenix Central Laboratory for completeserum chemistry panels. To assess myeloid:erythroid ratios, the bonemarrow from one femur was applied to CytoSpin slides (CYTOSPIN 3CYTOCENTRIFUGE and CYTO SLIDES, Shandon, Pittsburgh,Pa.) and sent toPhoenix Central Laboratories for analysis.

Study Results

There were no apparent clinical indications of physiologic effects or oftoxicity of human IL-21 at doses of 50 μg/day or lower. Body weights andtemperatures remained normal for the duration of the treatments. Serumchemistry parameters were in normal ranges. Red blood cell and plateletcounts appeared normal. In the mice receiving 50 μg/day for 8 days,manual differential white blood cell counts showed that the percentageof monocytes was elevated in the peripheral blood, and an apparentincrease in the total white blood cell counts. In bone marrow flushedfrom a femur, myeloid to erythroid ratios were increased in the 50 μgdose group, and to a lesser degree the 5 μg dose group from the 8-daydose set. In a non-parametric multiple column comparison using InStat(InStat MAC; GraphPad Software, Inc., San Diego, Calif.), thisdifference was statistically significant (p=0.0049). The differencebetween the highest dose group and vehicle was also significant,(p=0.0286). The increased white blood cells in peripheral blood and thesignificant increase in myeloid precursors in the marrow may thus berelated.

Histologic evaluation of the following tissues showed no apparentevidence of cytologic or structural changes, mitotic events or necrosis:thymus, liver, kidney, adrenal gland, duodenum, pancreas, jejunum,caecum, colon, mesenteric lymph nodes, uterus, ovary, salivary gland,heart, trachea, lung, and brain. There were no apparent differencesbetween the treatment groups in the weights of the thymus, kidney, liveror brain. Of all the tissues examined, only the spleen weights weresignificantly affected.

Each mouse spleen weight was normalized to her brain weight. In the 50μg/day treatment group compared to the vehicle, 0.1 μg and 0.5 μgtreatment groups, the average of the spleen weights was nearly 50%greater after four days of treatment and almost 100% greater after eightdays than the average spleen weights of the other three groups. In thefour-day set, the 5 μg/day group also tended to have larger spleens thanthe control and low dose groups. The difference in the spleen/brainweights with data from the four-day and the eight-day sets combined bytreatment group was statistically significant (p=0.0072) byKruskall-Wallace non-parametric ANOVA, multiple column comparison testusing the InStat program (GraphPad Software).

A marginal increase in extrameduallary hematopoiesis, especially in thered pulp was seen in spleens of mice from the highest dose group, evenin the mice treated for four days. Flow cytometric analysis of thespleens showed a significant increase in the proportion of myeloid sizecells in the highest dose group (p=0.01, Student's t test), representingincreases in both monocytes and neutrophils. This effect may be relatedto the increased peripheral blood mononuclear cell percentage, as wellas the apparent increase in myeloid precursors in the bone marrow,described above. Moreover, the transgenic mice derived from insertion ofthe human zalpha11 gene had increased extramedullary hematopoiesis intheir spleens compared to non-transgenic litter mates.

Several changes were observed in the 50 μg per day dose group comparedto the control group that implicate IL-21 in production or developmentof cells of the myeloid lineage. Taken together, the observed changessuggest that zalpha11 may be useful as a therapeutic protein in suchmedical specialties as cancer and immunologic disorders describedherein.

Example 4

Preliminary Elimination and Tissue Distribution Study Of PurifiedRecombinant Human IL-21 Protein

A. Summary

In order to elucidate tissue distribution and elimination patterns ofthe purified rhIL-21, a preliminary pharmacokinetic study wasundertaken. Nine week old male C57B1/6 mice were given purifiedrecombinant human IL-21 protein labeled with ¹¹¹Indium (¹¹¹In) (NEN,Boston, Mass.) by one of three routes. A single bolus injection wasgiven to each mouse by either the intravenous (IV), intraperitoneal(IP), or subcutaneous route (SC). The mice injected by either thesubcutaneous or intraperitoneal route were sacrificed at either one orthree hours after injection. The mice injected intravenously weresacrificed after either ten minutes or one hour following injection.Blood, plasma and selected tissues were harvested at various timepointsand counted by a gamma counter to estimate the approximate half-life andtissue distribution of the exogenous labeled protein. The tissues thatwere harvested for counting as well as the intervals of sacrifice wereselected based on reports of the distribution of other cytokines labeledwith radionuclides.

At sacrifice, tissues harvested for counting of radioactivity includedthymus, spleen, kidney, a lobe of liver, a lobe of lung, and urinarybladder. In the group receiving the injection intraperitoneally, gut wasalso counted to assess incidence of injection into the gut, and in thesubcutaneously dosed mice, skin with underlying structures in the areaof injection was counted. The cpm for whole liver and lung werecalculated from a section that was counted and a percentage of the wholeorgan weight represented by the section.

After the end of the study the collected tissues, whole blood and plasmawere counted on the COBRA II AUTO-GAMMA® gamma counter (PackardInstrument Company, Meriden, Conn.). An aliquot of the original labeleddosing solution was also counted at the end of the study with thetissues. This allowed calculation of percent total injectedradioactivity for each mouse and simultaneous correction of all countsfor radioactive decay. Approximations of remaining blood volume andorgan weights indicated that the majority of the counts administeredwere accounted for, and therefore the percentage of counts per tissuewere a reasonable representation of distribution of the counts followinglabeled IL-21 administration by each route.

B. ¹¹¹Indium Labeling of IL-21

Purified recombinant human IL-21 (U.S. Pat. No. 6,307,024) wasconjugated with a 10 fold molar excess of DTPA (Peirce, Rockford, Ill.)by incubating 30 minutes at room temperature in PBS. Unreacted DTPA andhydrolyzates were removed by buffer exchange on a Biomax-5k NMWL(Ultrafree-15, Millipore, Bedford, Mass.). The void volume protein peakwas concentrated to 5 mg/ml and an aliquot taken for testing in abioassay (anti-CD40 stimulation of murine B-cells (Example 10)). Uponconfirming that the DTPA-conjugate still had full bioactivity theconjugate was diluted to 0.5 mg/ml with 1M Na Acetate pH 6.0. Two mCi of¹¹¹Indium was taken up in 0.5 ml 1M Na Acetate pH 6.0 and mixed with theDTPA-human IL-21 for 30 min. at room temperature. Unincorporated¹¹¹Indium was removed during buffer exchange to PBS on a PD-10 column(Pharmacia, Piscataway, N.J.). The radio-labeled material was dilutedwith unlabeled human IL-21 to give a specific activity of 100 mCi/mg,sterile filtered and stored at 4° C. overnight. One hundred percent ofthe labeled protein was retained on a Biomax-5k NMWL membrane(Millipore). The labeled ¹¹¹In-human IL-21 was administered to mice inthe elimination and pharmacokinetic studies. Fifty μg human IL-21protein labeled with 5 μCi of labeled human IL-21 in 0.1 ml of PBSvehicle was administered to each animal.

C. Results Of Preliminary Distribution Study

After one and three hours following administration by all three routes,the highest concentration of ¹¹¹In-human IL-21, was found in kidney andthe second highest was in urine and urinary bladder, as evinced by thesetissues having the highest cpm. The average counts recovered fromkidneys were from 3 to 8 times higher than the whole liver counts,depending on the route of injection and the sacrifice timepoint. Forexample, the average kidney cpm at 60 minutes following IV injection was4.5 times greater than the average counts calculated for whole liverfrom the same group. In the group that was sacrificed ten minutes afterintravenous administration, the highest cpm was again in kidney, and thesecond highest accumulation was equivalent in liver, urinary bladder andurine.

D. Preliminary Pharmacokinetic Study

Blood and plasma collections were done at 10, 30 and 60 minutesfollowing injection by all three routes. Following injection by the IVroute, a separate set of mice had blood and plasma samples taken at two,five and ten minutes. Another set of mice who received their injectionsby either the IP or SC route had blood sampled at one, two and threehours. For the treatment groups see Table 4. The short collection timesbracket the reported half-life of IL-2 following intravenous injection.The reported T½ was in the range of 2.5 to 5.1 minutes. For reference toin vivo administration to IL-2, see Donohue J H and Rosenberg S A JImmunol 130:2203, 1983. The long timepoints were chosen to outline theanticipated elimination phase. TABLE 4 Route of injection Bleed Times(min.) Sacrifice Time Intravenous Group 1  2, 5, 10 10 min. IntravenousGroup 2 10, 30, 60 60 min. Intraperitoneal Group 1 10, 30, 60 60 min.Intraperitoneal Group 2 60, 120, 180 180 min.  Subcutaneous Group 1 10,30, 60 60 min. Subcutaneous Group 2 60, 120, 180 180 min. 

Un-labeled IL-2 has been shown to be eliminated from the serum with ahalf-life of approximately three minutes in mice after IV injection. Forreference see Donahue, J H and Rosenburg supra. Following IP and SCinjection of similar amounts of IL-2, the duration of persistence ofIL-2 activity in serum was prolonged from 2 units/ml for less than 30minutes following IV injection to greater than 2 units/ml for 2 hoursfollowing IP and 6 hours following SC injections. The principle route ofclearance of IL-2 appears to be the kidney. IL-21 has been shown to bestructurally similar to IL-2, as discussed herein. Preliminaryevaluation of the elimination of IL-21 appears to be consistent with theapparent clearance of IL-2 by the kidneys, based on the accumulation ofcpm predominantly in the kidneys, followed by the urinary bladder andurine in the present study.

Estimations were made of pharmacokinetic parameters based on noncompartmental analysis of the cpm data obtained from the plasma, usingthe PK analysis program WinNonLin, Version 1.1, (Scientific ConsultingInc., Cary, N.C.). Plasma half-lives of IL-21 were estimated using thepredicted terminal elimination rate constants for intravenous,subcutaneous, and intraperitoneal administration of a 50 μg dose. Thepharmacokinetic results were estimations due to limited data points inthe terminal elimination region of the plasma concentration vs. timeprofiles. Moreover, the fit of the terminal elimination phase for SC andIP dosing required use of data from timepoints during which absorptionof the ¹¹¹In-human IL-21 was apparently still occurring. However,estimations of half-lives following intravenous, subcutaneous, andintraperitoneal dosing were 13.6 min., 18.8 min., and 34.3 min.,respectively. Since a dosing range was not evaluated it was not apparentwhether saturable or active elimination (Michaelis Menten kinetics) wasoccurring. Therefore, these half-life calculations are estimations.

Estimates of the bioavailability of the labeled protein were made basedon the area under the curve (AUC) following subcutaneous orintraperitoneal dosing compared to that of intravenous dosing. Theestimated bioavailability following subcutaneous and intraperitonealinjection were 35.8% and 63.9% respectively. Because only one proteindose was studied, the bioavailability was not evaluated as a function ofdose. The estimated clearance and volume of distribution (based on thedata from the intravenous injection) were 0.48 ml/min. and 6.1 ml,respectively.

Although the data are preliminary, the fate of IL-21 administered IV wassimilar to that reported for IL-2, another 4-helix bundle cytokine(Donahue, J H and Rosenburg, S A supra.). Like IL-2, IV-administeredIL-21 had a plasma half life of only minutes with the main clearance inthe kidney. Three hours after injection, the majority of the labeledmaterial extracted from kidney was still retained in a Biomax 5K NMLWmembrane (Millipore). Since it has previously been reported that theindium remains associated with protein even during lysosomal degradation(Staud, F. et al., J. Pharm. Sciences 88:577-585, 1999) IL-21 isaccumulating and may be degraded in the kidney. The current study alsoshowed, as observed with many other proteins, including IL-2 (Donahue, JH and Rosenburg, S A, supra.), that IP and SC administrationsignificantly prolonged the plasma levels of IL-21.

Example 5

Isolation and Expansion of Fresh Human Bone Marrow MNC CD34+ FractionUsing IL-21 for Assessment of NK Activity

A. Selection and Isolation of CD34+ Cells from Human Bone Marrow

Fresh human bone marrow mononuclear cells (MNC) were prepared to enrichfor cells having NK cell activity. Fresh human MNCs were obtained fromPoeitic Technologies (Gaithersburg, Md.). 10 ml alpha MEM (JRH, Lenexa,Kans.) containing 10% HIA FBS (Hyclone, Logan, Utah) and the antibiotic1% PSN (Gibco, BRL, Grand Island, N.Y.) was added to the cell suspensionand the cells were passed through a 100 μm sieve. The cells were thencounted, pelleted, washed with 10 ml PBS containing 2% FBS, thenpelleted again and resuspended in 1 ml PBS containing 2% FBS. Cellshaving a CD34 cell surface marker (CD34+ cells) were magneticallyseparated using a Detachabead kit with Dynabeads M-450 CD34 ((Dynal,Oslo, Norway), as per manufacturer's instructions. Both the CD34+ celland the CD34− cell fractions were further analyzed below.

B. Expansion of CD34+ Cells Using IL-21

A CD34+ cell fraction was plated into four wells in a 24-well plate.50,000 positively selected cells suspended in 1 ml Alpha MEM (JRH)containing 10% HIA FBS (Hyclone) and 1% PSN (Gibco/BRL), plus thevarious cytokines described below were plated in each of the 4 wells(1-4). Various reagents were used to test for IL-21-induced expansion ofthe CD34+ selected bone marrow MNCs: Reagents included human flt3 (R&D,Minneapolis, Minn.); purified human IL-21 (U.S. Pat. No. 6,307,024);human IL-15 (R&D). Reagents were combined as follows at day 0: In well#1, 2 ng/ml human flt3 was added. In well #2, 2ng/ml human flt3 and 15ng/ml purified human IL-21 were added. In well #3, 2 ng/ml human flt3and 20 ng/ml human IL15 were added. In well #4, 2 ng/ml human flt3, 15ng/ml purified human IL-21, and 20 ng/ml human IL15 were added. Afterincubating for 18 days, the suspension cells from each well werepelleted, and then resuspended in 0.5 ml alpha MEM (JRH) containing 10%HIA FBS (Hyclone) and 1% PSN (Gibco/BRL), and counted to assessproliferation of the CD34+ cell fraction. A low level of proliferationwas seen in the presence of flt3 alone (control well #1), but thepresence of IL-15 or IL-21 in addition to flt3 had not significanteffect on the expansion (wells, #2 and #3). However, expansion beyondthe flt3 control was evident in well #4 which contained IL-15 and IL-21in addition to flt3. This result suggested that IL-21 and IL-15 act insynergy to expand the human CD34+ cell population. Moreover, the resultsof this experiment supported the results seen with the mouse IL-21 inthe mouse BM assay (Example 1).

All cell populations were then tested for NK activity and subjected toflow cytometry analysis, as shown below (Example 7).

C. Expansion of CD34+ or CD34− Cells Using IL-21 with Delayed Additionof IL-15

Both CD34 positive and negative (CD34−) fractions were plated separatelyinto six 12 well plate wells (1-6). Each of six wells contained 100,000positively or negatively selected cells in 2 ml alpha MEM containing 10%HIA FBS and PSN, described above. Reagents used were as described above.In well #1, 2 ng/ml human flt3 was added at day 0. In well #2, 2 ng/mlhuman flt3 was added at day 0, and after 5 days incubation 20 ng/mlhuman IL15 was added. In well #3, 2 ng/ml human flt3 and 15 ng/ml humanIL-21 were added at day 0. In well #4, 2 ng/ml human flt3 and 15 ng/mlhuman IL-21 were added at day 0, and after 5 days incubation 20 ng/mlhuman IL15 was added. In well #5, 2 ng/ml human flt3 and 20 ng/ml humanIL15 were added at day 0. In well #6, 2 ng/ml human flt3, 15 ng/ml humanIL-21, and 20 ng/ml human IL15 were added at day 0. After incubating fora total of 15 days from the start of the experiment, the cells from eachwell were harvested and counted.

In the CD34+ population a low level of proliferation was seen in thepresence of flt3 alone (control well #1), but the presence of IL-15 orIL-21 added at day 0 in addition to flt3 had no significant effect onthe expansion (wells, #3 and #5). Addition of IL-15 after 5 days hadsome proliferative effect in comparison to the flt3 control (well #2compared to well #1) and a proliferative effect in the presence ofzalpha11 (well #4 compared to well #3). However, the greatest expansionwas evident in well #6 which contained IL-15 and IL-21 in addition toflt3 at day 0.

In the CD34− population, no proliferation was seen in the presence offlt3 alone (control well #1), and in fact a decrease in the cellpopulation was evident. The presence of zalpha11 added at day 0 inaddition to flt3 (well #3) was similar to the flt3 control. The presenceof IL-15 added at day 5 increased proliferation effect of the cells inthe presence (well #4) or absence (well #2) of IL-21. Again, thegreatest expansion was evident in well #6 which contained IL-15 andIL-21 in addition to flt3 at day 0.

All cell populations were then tested for NK activity and subjected toFACS analysis, as shown below (Example 7).

Example 6

Isolation and Expansion of Fresh Mouse Cells Using Human and Mouse IL-21for Assessment of NK Activity and NK Cell Markers

A. Isolation and Expansion of Fresh Mouse Low Density Bone Marrow CellsUsing Human and Mouse IL-21

Fresh mouse marrow cells were isolated by clipping both ends of mousefemurs, and flushing two to three milliliters of growth medium (seebelow) through the inside of the bone into a collection tube. The growthmedium was 500 ml RPMI 1640 Medium (JRH Biosciences. Lenexa, Kans.); 5ml 100× L-glutamine (Gibco BRL. Grand Island, N.Y.); 5 ml 100× NaPyruvate (Gibco BRL); 5 ml 100× Penicillin, Streptomycin, Neomycin (PSN)(Gibco BRL); and 50 ml heat-inactivated Fetal Bovine Serum (FBS)(Hyclone Laboratories. Logan, Utah). The marrow cells were thenbroken-up by pipeting the media up and down several times. The cellswere then pelleted and washed once with growth medium, and passedthrough a 70-micron sieve. The low-density mononuclear cells were thenisolated by subjecting the marrow cells to a density gradient. Marrowcells in five to eight milliliters of growth medium were carefullypipetted on top of five to eight milliliters of NycoPrep 1.077 Animal(Nycomed. Oslo, Norway) in a centrifuge tube. This gradient was thencentrifuged at 600 ×g for 20 minutes. The low density mononuclear cellswere harvested from the interface layer between the NycoPrep and themedium. These cells were then diluted to approximately 20 milliliters ingrowth medium, pelleted and washed. The cells were then plated atapproximately 0.5-1.5×10⁶ cells per milliliter in growth medium in astandard tissue culture flask and incubated at 37° C., 5% CO₂ for twohours.

The non-adherent, low density (NA LD) marrow cells were then harvestedand plated at 0.5-2.0×10⁵ cells per milliliter in growth medium plus 2.5nanograms per milliliter mouse flt3 (R and D Systems. Minneapolis,Minn.) plus 25 to 50 nanograms per milliliter human Interleukin 15(IL-15) (R and D Systems) with or without 50 to 150 nanograms permilliliter human IL-21; or with or without 0.12 to 10 nanograms permilliliter mouse IL-21.

There was no significant expansion without the addition of the human ormouse IL-21. Non-adherent cells were expanded in the cultures containingmouse IL-21 as low as 0.12 ng/ml and in the cultures containing humanIL-21 as low as 22 ng/ml. In cultures containing both the human andmouse IL-21, non-adherent cell expansion increased with increasing doseif IL-21, with the mouse ligand saturating response at about 5-10 ng/mland the human not reaching a saturating response even at the highestdose of 200 ng/ml. Human IL-21 appeared to be approximately 20 to 100fold less potent on mouse cells as the mouse IL-21. After approximatelyfive to ten days the IL-21 expanded mouse cells were harvested andanalyzed by flow cytometry (FACSCalibur; Becton Dickinson, Mansfield,Mass.) to determine what percentage of them were positive for NK cellantigens, where 46% were positive for the PanNK cell marker DX5(Pharmingen).

B. Isolation and Expansion of Fresh Lineage Depleted Mouse Marrow Cells

Fresh mouse lineage depleted (lin−) marrow cells were isolated fromfresh mouse marrow cells by first incubating the cells with thefollowing antibodies: TER119, Gr-1, B220, MAC-1, CD3e and I-Ab(Pharmingen. San Diego, Calif.). The lin+ cells were then removed withDynabeads M-450 sheep anti-rat IgG (Dynal, Lake Success, N.Y.) as permanufacturer's instructions.

The negatively selected lin- marrow cells were then plated as above ingrowth medium plus either 2.5 ng/mL flt3 (R&D Systems) and 25 ng/mLIL-15 (R&D Systems); or flt3, IL-15 and mouse IL-21, 2 to 5% BHK mouseIL-21 conditioned medium. After six days of growth, the cultures wereharvested, counted and submitted to an NK cell activity assay (Example7). Cells grown with mouse IL-21 were approximately two to three timesmore effective at lysing NK cell target cells (YAC-1 cells) as the cellsgrown without IL-21.

C. Isolation and Expansion of CD4− CD8− (Double Negative or DN)Thymocytes

Fresh mouse thymocytes were isolated by chopping and sieving thymusesfrom three to eight week old mice. CD4− CD8− (DN) cells were thennegatively selected by incubating the thymocytes with anti-CD4 andanti-CD8 antibodies (PharMingen), then removing the CD4+ CD8+ cells withDynabeads M-450 sheep anti-rat IgG (Dynal) as per manufacturer'sinstructions.

The DN mouse thymocytes were then grown in growth medium plus 2.5 ng/mLflt3 (R&D Systems), 25 ng/mL IL-15 (R&D Systems) and 10 ng/mL IL-7 (R&DSystems) with or without mouse IL-21 as above. Six days later the cellswere harvested, counted, analyzed by flow cytometry as described above,and also submitted to an NK cell activity assay (Example 7).

The culture grown with mouse IL-21 yielded approximately 480,000 cellswhile the culture without IL-21 yielded only approximately 160,000cells. The culture grown with mouse IL-21 was found to be approximately16.2% positive for the NK cell antigen Pan NK, DX5 (PharMingen). Theculture grown without IL-21 was 14.6% positive for DX5. The cells grownwith IL-21 lysed NK cell target cells, YAC-1, approximately two timesbetter than the cells grown without IL-21. The expanded cells did notlyse significantly a negative control target cell line, EL4. Theseresults suggested that IL-21 selectively expands lytic NK cells.

Example 7

Activity of Human and Mouse IL-21 Expanded Cells and Mature Murine NKCells in NK Cell Cytotoxicity Assays

A. NK Cell Assay

NK cell-mediated target cytolysis was examined by a standard⁵¹Cr-release assay. Target cells (K562 cells (ATCC No. CCL-243) in humanassays, and YAC-1 cells (ATCC No. TIB-160) in mouse assays) lackexpression of major histocompatability complex (MHC) molecules,rendering them susceptible to NK cell-mediated lysis. A negative controltarget cell line in mouse assays is the MHC⁺ thymoma EL4 (ATCC No.TIB-39). We grew K562, EL4, and YAC-1 cells in RP10 medium (standardRPMI 1640 (Gibco/BRL, Grand Island, N.Y.) supplemented with 10% FBS(Hyclone, Logan, Utah), as well as 4 mM glutamine (Gibco/BRL), 100I.U./ml penicillin+100 MCG/ml streptomycin (Gibco/BRL), 50 μMβ-mercaptoethanol (Gibco/BRL) and 10 mM HEPES buffer (Gibco/BRL). On theday of assay, 1-2×10⁶ target cells were harvested and resuspended at2.5-5×10⁶ cells/ml in RP10 medium. We added 50-100 μl of 5 mCi/ml⁵¹Cr-sodium chromate (NEN, Boston, Mass.) directly to the cells andincubated them for 1 hour at 37° C, then washed them twice with 12 ml ofPBS and resuspended them in 2 ml of RP 10 medium. After counting thecells on a hemacytometer, the target cells were diluted to 0.5-1×10⁵cells/ml and 100 μl (0.5-1×10⁴ cells) were mixed with effector cells asdescribed below.

In human assays, effector cells were prepared from selected and expandedhuman CD34⁺ BM cells (Example 5B) which were harvested, washed, counted,mixed at various concentrations with ⁵¹Cr-labeled target cells in96-well round bottomed plates, and incubated for 4 hours at 37° C. Afterco-incubation of effector cells and the labeled target cells, half ofthe supernatant from each well was collected and counted in a gammacounter for 1 min/sample. The percentage of specific ⁵¹Cr release wascalculated from the formula 100×(X−Y)/(Z−Y), where X is ⁵¹Cr release inthe presence of effector cells, Y is the spontaneous release in theabsence of effectors, and Z is the total ⁵¹Cr release from target cellsincubated with 0.5% Triton X-100. Data were plotted as the % specificlysis versus the effector-to-target ratio in each well.

B. Activity of Human IL-21 Expanded Cells

Isolated CD34+human HPCs cultured with flt3±IL-21 and flt3+IL-15±IL-21(Example 5), were harvested the cells on day 15 to assess their capacityto lyse MHC K562 cells in a standard ⁵¹Cr-release assay as describedabove, and to analyze their surface phenotype by flow cytometry. Asexpected from previous reports (Mrozek, E et al., Blood 87:2632-2640,1996; and Yu, H et al., Blood 92:3647-3657, 1998), simultaneous additionof IL-15 and flt3L did induce the outgrowth of a small population ofCD56+cells. Interestingly, although BM cells cultured simultaneouslywith IL-21 and flt3L did not expand significantly, there was asignificant increase in total cell numbers in cultures containing acombination of flt3L, IL-21 and IL-15 (see, Example 5).

For an assessment of the surface phenotype of these human BM cultures,we stained small aliquots of the cells for 3-color flow cytometricanalysis with anti-CD3-FITC, anti-CD56-PE and anti-CD16-CyChrome mAbs(all from PharMingen, San Diego, Calif.) and analyzed them on aFACSCalibur using CellQuest software (Becton Dickinson, Mountain View,Calif.). This flow cytometric analysis confirmed that the cells growingout of these cultures were differentiated NK cells, as they were largeand granular and expressed both CD56 and CD16, and were CD3 (Lanier, L LAnnu. Rev. Immunol. 16:359-393, 1998). Furthermore, these cellsexhibited significantly higher effector function than those cells grownwith IL-15 and flt3. More specifically, cells grown in all threecytokines lysed more than 40% of the K562 targets at aneffector-to-target ratio (E:T) of 1.5, whereas cells grown inIL-15+flt3L lysed fewer than 5% of the targets at an E:T of 2. Thesedata demonstrate that, in combination with IL-15, IL-21 stimulates thedifferentiation of NK cells from CD34⁺ BM cells.

C. Activity of Mouse IL-21 Expanded Cells

To test the effects of IL-21 on murine hematopoietic progenitor cells,purified Lineage-negative (Lin−) bone marrow cells from C57B1/6 micewere expanded in flt3+IL-15±IL-21, as described in Example 6B. On day 6of culture, the cells (“effectors”) were harvested and counted, thenresuspended in 0.4 ml of RP10 medium (Example 7A). Two aliquots (0.15 mleach) of each sample expanded with or without IL-21 (Example 7A) werediluted serially 3-fold in duplicate in 96-well round bottomed plates,for a total of 6 wells of 100 μl each. The remaining 100 μl of cellswere stained for NK cell surface markers with FITC-anti-2B4 andPE-anti-DX5 mAbs (PharMingen) and analyzed by flow cytometry. Each groupof cells exposed to flt3+IL-15 with or without the presence of IL-21 hadsimilar fractions of 2B4+DX5+cells, ranging from 65-75% positive forboth NK markers.

For the NK lysis assay, target cells (YAC-1 and EL4) were labeled with⁵¹Cr as described above. After counting the target cells on ahemacytometer, the target cells were diluted to 0.5-1×10⁵ cells/ml and100 μl of YAC-1 or EL4 (0.5-1×10⁴ cells) were mixed with 100 μl effectorcells and incubated for 4 hours at 37° C. Specific lysis was determinedfor each well as described above.

We found that cells grown in the presence of flt3+IL-15+IL-21 exhibitedenhanced lytic activity (roughly 2-fold) against the YAC-1 targets (butdid not kill the MHC⁺ control cell line EL4). At an effector-to-targetratio (E:T) of 5, NK cells generated in the presence of all 3 cytokines(IL-21+flt3+IL-15) lysed 12% of the YAC-1 cells, whereas those NK cellsexpanded with flt3+IL-15 lysed 6% of the YAC-1 targets. Subsequentexperiments confirmed this trend.

In a second approach to determine the biological activity of IL-21 onmurine NK cells, we isolated immature CD4⁻CD8⁻ (“double negative”, DN)mouse thymocytes as described in Example 6C and cultured them withIL-15+flt3+IL-7 or IL-15+flt3+IL-2, with or without IL-21. On day 6 ofculture, the cells were harvested and assayed for NK lytic activity onYAC-1 and EL4 cells as described above. We found that cells cultured inthe presence of IL-21 had the greatest lytic activity in this assay,with enhanced lytic activity over those cells cultured in the presenceof the other cytokines. Specifically, DN thymocytes grown withIL-15+flt3+IL-7 killed 18% of the YAC-1 cells at E:T of 24 while cellsgrown in the presence of IL-15+flt3+IL-7 plus IL-21 killed 48% of thetargets at the same E:T. DN thymocytes grown in IL-15+flt3+IL-2 killed15% of the YAC-1 targets at an E:T of 6, whereas cells grown with these3 cytokines and IL-21 killed 35% of the YAC-1 cells at an E:T of 9. Flowcytometry was performed on the cultured cells one day before the NKlysis assay. As was true for the bone marrow cultures, despite theproliferative effect of IL-21 (cell numbers increase approximately2-fold when IL-21 is added), it did not significantly enhance thefraction of DX5⁺ cells (17-20% of total cells in the cultures with IL-7,and 35-46% of total in cultures with IL-2). These data imply that IL-21,in combination with IL-15 and flt3, enhances the lytic activity of NKcells generated from murine bone marrow or thymus.

D. Activity of Mouse IL-21 on Mature Murine NK Cells

In order to test the effects of mouse IL-21 on mature NK cells, weisolated spleens from four 5-week old C57B1/6 mice (JacksonLaboratories, Bar Harbor, Me.) and mashed them with frosted-end glassslides to create a cell suspension. Red blood cells were removed byhypotonic lysis as follows: cells were pelleted and the supernatantremoved by aspiration. We disrupted the pellet with gentle vortexing,then added 900 μl of sterile water while shaking, followed quickly (lessthan 5 sec later) by 100 μl of 10× HBSS (Gibco/BRL). The cells were thenresuspended in 10 ml of 1× HBSS and debris was removed by passing thecells over a nylon mesh-lined cell strainer (Falcon). These RBC-depletedspleen cells were then pelleted and resuspended in MACS buffer (PBS+1%BSA+2 mM EDTA) and counted. We stained 300×10⁶ of the cells withanti-DX5-coated magnetic beads (Miltenyi Biotec) and positively selectedDX5⁺ NK cells over a MACS VS+ separation column, according to themanufacturer's instructions, leading to the recovery of 8.4×10⁶ DX5⁺cells and 251×10⁶ DX5⁻ cells. Each of these groups of cells werecultured in 24-well plates (0.67×10⁶ cells/well, 2 wells per treatmentcondition) in RP10 medium (Example 7A) alone or with 1) 30 ng/ml mouseIL-21, 2) 30 ng/ml recombinant mouse IL-2 (R&D Systems, Inc.,Minneapolis, Minn.), 3) 30 ng/ml recombinant human IL-15 (R&D), 4) 30ng/ml each of mouse IL-21 and hIL-15, or 5) 30 ng/ml each of mIL-2 andhIL-15. The cells were harvested after 21 hours, washed, and resuspendedin RP10 medium and counted. The cells were then assayed for theirability to lyse ⁵¹Cr-labeled YAC-1 or EL4 targets cells, as described inExample 7A.

In general, there was little NK activity from the DX5⁻ (non-NK cells)groups, but the DX5⁻ cells cultured with IL-21 and hIL-15 did lyse 25%of the YAC-1 target cells at an E:T of 82. By comparison, DX5⁻ cellscultured with hIL-15 alone lysed 14% of the YAC-1 targets at an E:T of110. This suggests that IL-21 and IL-15 are acting together on theresidual NK1.1⁺ NK cells in this cell preparation. As for the DX5⁺ cellpreparation, treatment with mouse IL-21 alone did not significantlyincrease their effector function (their lysis of YAC-1 cells was similarto the untreated group). As expected, both IL-2 and IL-15 significantlyimproved NK activity. The highest level of lysis, however, was detectedin the group treated with IL-21 and hIL-15 (65% lysis of YAC-1 cells atan E:T of 3.3, vs. 45% lysis at an E:T of 4 for the hIL-15 treatmentgroup). Taken together, these results suggest that although IL-21 alonemay not increase NK cell lysis activity, it does enhance NK lysisactivity of mature NK cells, when administered with IL-15.

Example 8

IL-21 Proliferation of Human and Mouse T-cells in a T-cell ProliferationAssay

A. Murine IL-21 Proliferation of Mouse T-cells

T cells from C57B1/6 mice (Jackson Laboratories, Bar Harbor, Me.) wereisolated from pooled splenocytes and lymphocytes from axillary,brachial, inguinal, cervical, and mesenteric lymph nodes (LNs). Spleenswere mashed with frosted-end glass slides to create a cell suspension.LNs were teased apart with forceps and passed through a cell strainer toremove debris. Pooled splenocytes and LN cells were separated into CD8⁺and CD4⁺ subsets using two successive MACS magnetic separation columns,according to the manufacturer's instructions (Miltenyi Biotec, Auburn,Calif.). Whole thymocytes were collected from the same mice.

Cells were cultured at 3×10⁵ cells/well (thymocytes) or 10⁵ cells/well(mature T cells) with increasing concentrations of purified murine IL-21(0-30 ng/ml) (U.S. Pat. No. 6,307,024) in 96-well flat bottomed platespre-coated overnight at 4° C. with various concentrations of anti-CD3mAb 2C11 (PharMingen) for 3 days at 37° C. The anti-CD3 antibody servedto activate the murine T-cells through the T-cell receptor. Each wellwas pulsed with 1 μCi ³H-thymidine on day 2 and plates were harvestedand counted 16 hours later to assess proliferation.

When we tested IL-21 in T cell proliferation assays, we found that itco-stimulated anti-CD3-activated murine thymocytes, leading to anaccelerated outgrowth of CD8⁻CD4⁻ cells (the majority of the thymocytescultured with anti-CD3+IL-21 were CD8CD4⁻ by day 3 of culture, whilecells cultured with anti-CD3 alone did not significantly skew to thisphenotype until day 5). We did not observe significant levels ofproliferation of thymocytes to IL-21 in the absence of anti-CD3.

Interestingly, when we assayed mature peripheral murine T cells fortheir ability to respond to IL-21+anti-CD3, we found that only the CD8⁺,but not the CD4⁺ subset, responded in a dose-dependent manner to IL-21.We also observed weak but reproducible proliferation of CD8⁺ cells (butnot CD4⁺ cells) in response to IL-21 alone. Interestingly, this was notobserved for human T cells (see Example 8B, below).

B. Human IL-21 Proliferation of Human T-cells

Human CD4⁺ and CD8⁺ T cells were isolated from PBMC as described inExample 9 (below) Cells were cultured at about 10⁵ cells/well withincreasing concentrations of purified human IL-21 (0-50 ng/ml) (U.S.Pat. No. 6,307,024) in 96-well flat bottomed plates pre-coated overnightat 4° C. with various concentrations of anti-human CD3 mAb UCHT1(PharMingen) for 3 days at 37° C. Each well was pulsed with 1 uCi³H-thymidine on day 2 and plates were harvested and counted 16 hourslater. Unlike our results with mouse T cells, our preliminary datasuggests that human IL-21 co-stimulates CD4+, but not CD8+, human Tcells in a dose-dependent fashion.

In other experiments, mature murine CD4+ and CD8+ T cells were enrichedfrom pooled C57B1/6 spleen and LN cells by depletion of CD19⁺ B cellsusing a magnetic bead column. The resulting cell populations wereassayed for proliferation to plate-bound anti-mouse CD3ε mAb in theabsence or presence of increasing concentrations of murine IL-21, asindicated. Data shown are representative of results from 4 experiments.

T cells from C57B1/6 mice were isolated from pooled splenocytes andlymphocytes from auxiliary, brachial, inguinal, cervical, and mesentericLNs. Spleens were mashed with frosted-end glass slides to create a cellsuspension. LNs were teased apart with forceps and passed through a cellstrainer to remove debris. Pooled splenocytes and LN cells wereseparated into CD8⁺ and CD4⁺ subsets using two successive MACS magneticseparation columns, according to the manufacturer's instructions(Miltenyi Biotec, Sunnyvale, Calif.). Cells were cultured at 10⁵/wellwith increasing concentrations of murine IL-21 (0-30 ng/ml) in 96-wellflat bottomed plates pre-coated overnight at 4° C. with variousconcentrations of anti-CD3 mAb 2C11 (PharMingen) for 3 days at 37° C.Each well was pulsed with 1 μCi ³H-thymidine on day 2 and plates wereharvested and counted 16 hours later.

Table 5 illustrates that mIL-21 co-stimulates the proliferation ofmurine CD8+ T cells. Values represent the CPM incorporated of³H-thymidine (average±standard deviation of triplicate wells). TABLE 5Anti-CD3 mAb (ug/ml) ng/ml mIL-21 0 0.11 0.33 1.0 3.0 CD4+ 0 405 +/− 101 67895 +/− 18752 141175 +/− 6733 202251 +/− 35571 246626 +/− 45106 1.2247 +/− 86   80872 +/− 23598 126487 +/− 7472 178863 +/− 33583 205861 +/−14675 6 302 +/− 106 75192 +/− 5323 102005 +/− 20059 191598 +/− 15881218718 +/− 12142 30 364 +/− 126 86164 +/− 8065 141065 +/− 4921 186089+/− 17585 266650 +/− 39839 CD8+ 0 168 +/− 47  40198 +/− 4557  70272 +/−4141  84771 +/− 9450  97869 +/− 3368 1.2 268 +/− 117 50095 +/− 3959 84319 +/− 6373 105176 +/− 10828 113394 +/− 3657 6 323 +/− 159 78113 +/−6967 108461 +/− 2175 132301 +/− 13386 178551 +/− 16373 30 2007 +/− 470 132238 +/− 1915  182485 +/− 4991 272229 +/− 9325 330434 +/− 47185

Example 9

Real Time PCR Shows IL-21 Expression in Human CD4+ Cells

A. Purified Human T Cells as a Primary Source Used to Assess Human IL-21Expression

Whole blood (150 ml) was collected from a healthy human donor and mixed1:1 with PBS in 50 ml conical tubes. Thirty ml of diluted blood was thenunderlayed with 15 ml of Ficoll Paque Plus (Amersham Pharmacia Biotech,Uppsala, Sweden). These gradients were centrifuged 30 min at 500 g andallowed to stop without braking. The RBC-depleted cells at the interface(PBMC) were collected and washed 3 times with PBS. The isolated humanPBMC yield was 200×10⁶ prior to selection described below.

The PBMCs were suspended in 1.5 ml MACS buffer (PBS, 0.5% EDTA, 2 mMEDTA) and 3×10⁶ cells were set aside for control RNA and for flowcytometric analysis. We next added 0.25 ml anti-human CD8 microbeads(Miltenyi Biotec) and the mixture was incubated for 15 min at 4° C.These cells labeled with CD8 beads were washed with 30 ml MACS buffer,and then resuspended in 2 ml MACS buffer.

A VS+ column (Miltenyi) was prepared according to the manufacturer'sinstructions. The VS+ column was then placed in a VanioMACS magneticfield (Miltenyi). The column was equilibrated with 5 ml MACS buffer. Theisolated primary mouse cells were then applied to the column. The CD8negative cells were allowed to pass through. The column was rinsed with9 ml (3×3 ml) MACS buffer. The column was then removed from the magnetand placed over a 15 ml falcon tube. CD8+ cells were eluted by adding 5ml MACS buffer to the column and bound cells flushed out using theplunger provided by the manufacturer. The yield of CD8+ selected humanperipheral T cells was about 51×10⁶ total cells. The CD8-negative flowthrough cells were collected, counted, stained with anti-human CD4coated beads, then incubated and passed over a new VS+ column at thesame concentrations as described above. The yield of CD4+ selected humanperipheral T cells was 42×10⁶ total cells.

A sample of each of the CD8+ and CD4+ selected human T cells was removedfor staining and sorting on a fluorescence activated cell sorter (FACS)to assess their purity. A PE-conjugated anti-human CD4 antibody, ananti-human CD8-FITC Ab, and an anti-human CD19-CyChrome Ab (all fromPharMingen) were used for staining the CD8+ and CD4+ selected cells. TheCD8-selected cells in this first experiment were 80% CD8+, and theCD4-selected cells were 85% CD4+. In 2 subsequent experiments (Example9B), the CD8+ purified cells were 84% and 81% pure, and the CD4+ cellswere 85% and 97% pure, respectively. In one experiment, we stained thenon-binding (flow-through) cells with anti-human CD19-coated beads(Miltenyi) and ran them over a third magnetic bead column to isolateCD19+ B cells (these were 92% pure).

The human CD8+, CD4+and CD19+ selected cells were activated byincubating 0.5×10⁶ cells/ml in RPMI+5% human ultraserum (GeminiBioproducts, Calabasas, Calif.)+PMA 10 ng/ml and Tonomycin 0.5 μg/ml(Calbiochem) for about 4, 16, or 24 hours at 37° C. The T-cells(2.5×10⁶/well) were alternately stimulated in 24-well plates pre-coatedovernight with 0.5 μg/ml plate-bound anti-CD3 mAb UCHT1 (PharMingen)with or without soluble anti-CD28 mAb (PharMingen) at 5 μg/ml. At eachtimepoint, the cells were harvested, pelleted, washed once with PBS, andpelleted again. The supernatant was removed and the pellets weresnap-frozen in a dry ice/ethanol bath, then stored at −80° C. for RNApreparation at a later date.

Real Time-PCR was performed on these human CD8+, CD4+and CD19+ selectedcells as described in Example 9B and Example 9C below for assessinghuman IL-21 and receptor expression.

B. Primers and Probes for Quantitative RT-PCR for Human IL-21 Expression

Real-time quantitative RT-PCR using the ABI PRISM 7700 SequenceDetection System (PE Applied Biosystems, Inc., Foster City, Calif.) hasbeen previously described (see, Heid, C A et al., Genome Research6:986-994, 1996; Gibson, U E M et al., Genome Research 6: 995-1001,1996; and Sundaresan, S et al., Endocrinology 139:4756-4764, 1998). Thismethod incorporates use of a gene specific probe containing bothreporter and quencher dyes. When the probe is intact the reporter dyeemission is negated due to the proximity of the quencher dye. During PCRextension using additional gene-specific forward and reverse primers,the probe is cleaved by 5′ nuclease activity of Taq polymerase whichreleases the reporter dye resulting in an increase in fluorescentemission.

The primers and probes used for real-time quantitative RT-PCR analyseswere designed using the primer design software Primer Express™ (PEApplied Biosystems). Primers for human IL-21 were designed spanning anintron-exon junction to eliminate amplification of genomic DNA. Theforward primer, ZC22,281 (SEQ ID NO: 12) and the reverse primer,ZC22,279 (SEQ ID NO12) were both used at 300 nM concentration tosynthesize an 80 bp product. The corresponding IL-21 TaqMan probe, ZG32(SEQ ID NO: 14) was synthesized by PE Applied Biosystems. The probe waslabeled with a reporter fluorescent dye (6-carboxy-fluorescein) (FAM)(PE Applied Biosystems) at the 5′ end and a quencher fluorescent dye(6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied Biosystems) at the3′ end. In order to test the integrity or quality of all the RNAsamples, they were screened for rRNA using the primer and probe setordered from PE Applied Biosystems (cat No. 4304483). The reporterfluorescent dye for this probe is VIC (PE Applied Biosystems). The rRNAresults will allow for the normalization of the IL-21 results.

RNA was prepared from pellets provided in Example 9A, using RNeasyMiniprep™ Kit (Qiagen, Valencia, Calif.) per the manufacturer'sinstructions. Control RNA was prepared from about 10 million BHK cellsexpressing human IL-21.

C. Primers and Probes for Quantitative RT-PCR for Human zalpha11Receptor Expression

Real time PCR was performed to assess the expression of IL-21 receptoras per Example 9B and Example 9D, using the cells prepared under theconditions detailed in 43A, and probes specific for the IL-21 receptor.The forward primer, ZC22,277 (SEQ ID NO:15) and the reverse primer,ZC22,276 (SEQ ID NO:16) were used in a PCR reaction (above) at about 300nM concentration to synthesize a 143 bp product. The corresponding IL-21TaqManQ probe, designated ZG31 (SEQ ID NO: 17) was synthesized andlabeled by PE Applied Biosystems. RNA from BaF3 cells expressing humanIL-21 receptor was used to generate appropriate control for standardcurves for the real-time PCR described in Example 9D below.

D. Real-time Quantitative RT-PCR

Relative levels of IL-21 RNA were determined by analysis of total RNAsamples using the One-Step RT-PCR method (PE Applied Biosystems). RNAfrom BHK cells expressing human IL-21 was used to generate a standardcurve. The curve consisted of serial dilutions ranging from 2.5-2.5×10⁴ng for the rRNA screen and 25-0.0025 ng for the IL-21 screen with eachpoint analyzed in triplicate. The total RNA samples were also analyzedin triplicate for human IL-21 transcript levels and for levels of rRNAas an endogenous control. Each One-step RT-PCR reaction consisted of 25ng of total RNA in buffer A (50 mM KCL, 10 mM Tris-HCL, and the internalstandard dye, ROX (PE Applied Biosystems)), appropriate primers (50 nMfor rRNA samples, 300 nM for IL-21 samples) and probe (50 nM for rRNA,100 nM for IL-21 ), 5.5 mM MgCl₂, 300 μM each d-CTP, d-ATP, and d-GTPand 600 μM of d-UTP, reverse transcriptase (0.25 U/μl), AmpliTaq DNApolymerase (0.025 U/μl) and RNase Inhibitor (0.4 U/μl) in a total volumeof 25 μl. Thermal cycling conditions consisted of an initial RT step at48° C. for 30 minutes, an AmpliTaq Gold activation step of 95° C. for 10minutes, followed by 40 cycles of amplification for 15 seconds at 95° C.and 1 minute at 60° C. Relative IL-21 RNA levels were determined by theStandard Curve Method as described in User Bulletin No. 2 (PEBiosystems; User Bulletin #2: ABI Prism 7700 Sequence Detection System,Relative Quantitation of Gene Expression, Dec. 11, 1997) using the rRNAmeasurements to normalize the IL-21 levels. Samples were comparedrelative to the calibrator within each experiment. The calibrator wasarbitrarily chosen based on good quality RNA and an expression level towhich other samples could significantly be compared. Results of theexperiments analyzing the expression of the IL-21 and IL-21 receptor instimulated and unstimulated cells (Example 9A) are as described inExample 9E below.

E. Expression of Human zalpha11 Receptor and Ligand in CD4+, CD8+ andCD19+ Cells

The first experiment used RT-PCR, described above, to assess zalpha11receptor expression in unstimulated and anti-CD3 stimulated CD4+ andCD8+ samples at timepoints of 0 h (unstimulated (“resting”) cells), andat 4 h, 15.5 h and 24 h, after stimulatoin. The resting CD4+ sample wasarbitrarily chosen as the calibrator and given a value of 1.00. Therewas approximately a 4-fold increase in receptor expression inunstimulated CD4+ cells from 4 h to 24 h of culture and about an 8-foldincrease over the same time period in anti-CD3 stimulated CD4+ cells.The CD8+ cells showed a 7-fold increase in IL-21 receptor expressionthat peaked at 4 hrs and decreased over time. With anti-CD3 stimulation,the CD8+ cells had a constant 8-fold increase in receptor expression.

This first experiment also used RT-PCR to assess IL-21 expression in thesame anti-CD3 stimulated and unstimulated CD4+ and CD8+ samples. The 4hr anti-CD3 stimulated CD8+ sample was arbitrarily chosen as thecalibrator and given a value of 1.00. The results showed thatunstimulated CD4+ and CD8+ cells do not express IL-21 . We observed asignificant elevation of expression in the anti-CD3 stimulated CD4+cells at 4 h, with about a 300-fold increase in signal observed at 15.5h. The CD8+ cells expressed a small amount of ligand upon anti-CD3stimulation, however this is probably due to contamination of the CD8+population with a small number of CD4+ cells.

The second experiment used RT-PCR to assess IL-21 receptor expression inanti-CD3-stimulated, PMA+Tonomycin-stimulated and unstimulated CD4+ andCD8+ samples at timepoints of 0 h, and at 3.5 h, 16 h and 24 h afteractivation. The resting CD8+ sample was arbitrarily chosen as thecalibrator and given a value of 1.00. The resting CD4+ and CD8+ cellsdid not have significant amounts of receptor expression. The expressionwas about 3 fold higher in the PMA+Tonomycin-stimulated CD4+ samples at3.5 h, 16 h and 24 h after stimulation. The expression in anti-CD3activated CD4+ cells peaked at 10-fold above background levels at 3.5 hafter stimulation, then fell back to levels 4-fold above background at16 h after stimulation. The CD8+ cells showed a 4-fold expressionincrease at 3.5 h after PMA+Tonomycin stimulation, with expressiondecreasing at subsequent timepoints. As in the first experiment, theanti-CD3 stimulated CD8+ cells again exhibited an 8-fold abovebackground induction of receptor expression.

These samples from the second experiment were also used to assess IL-21expression. The 24 hr PMA+Tonomycin stimulated CD4+ sample wasarbitrarily chosen as the calibrator and given a value of 1.00. Theresults showed that again none of the unstimulated cells expressedIL-21. There was about a 30-fold induction of ligand expression in theCD4+ cells stimulated with anti-CD3 at 3.5 h, as seen in the previousexperiment (at 4 h). However, there was only about a 5-fold inductionwith PMA+Tonomycin stimulation at 3.5 h that went down at subsequenttimepoints. Again, the CD8+ cells expressed a very small amount of IL-21that was probably attributed to contaminating CD4+ cells.

The final experiment used RT-PCR to assess IL-21 receptor expression inanti-CD3- and anti-CD3/anti-CD28-stimulated and unstimulated CD4+ andCD8+ samples at timepoints of 0 h, and at 2 h, 4 h, and 16 h afterstimulation. CD19+ cells activated with PMA+Ionomycin were also screenedfor receptor expression at the same time intervals. The resting CD4+sample was arbitrarily chosen as the calibrator and given a value of1.00. The 2 h anti-CD3 stimulated CD4+ cells only had a 4-fold inductionof receptor, compared to the 10-fold induction seen at 3.5 h in theprevious experiment. The combination of anti-CD3 and anti-CD28 increasedIL-21 receptor expression to 8-fold above background. The 16 hanti-CD3/anti-CD28 stimulated CD8+ cells had very low IL-21 receptorexpression levels, as seen in the CD8+ cells in previous experiments(above). The CD19+ cells stimulated with PMA+Ionomycin had the mostsignificant IL-21 receptor expression with a 19-fold increase at 2 h,but the expression levels decreased back to those of resting cells by 16h.

These samples from the final experiment were also used to assess IL-21by RT-PCR. The 16 h anti-CD3/anti-CD28 stimulated CD8+ sample wasarbitrarily chosen as the calibrator and given a value of 1.00. Theresults showed that at 2 h the CD4+ cells had about a 2-fold inductionof IL-21 expression with anti-CD3 stimulation and a 5-fold inductionwith anti-CD3 plus anti-CD28 stimulation. These stimulation conditionsinduced Ligand expression over time, with the 16 h stimulated CD4+ cellsexhibiting Ligand expression levels 70-fold above background. CD8+ andCD19+ cells showed no IL-21 expression.

A certain amount of variation was expected between blood draws (i.e.multiple samples at different times from the same patient and betweenmultiple patients). Therefore, data trends were analyzed within eachstudy or from a single blood sample and the three experiments above werecompared for an overall conclusion. The trend from the Real Time PCRexperiments described above is that of all the cell types tested, CD19+B cells activated with PMA+ionomycin expressed the highest levels ofIL-21 receptor RNA. CD4+ and CD8+ cells can also be stimulated toexpress receptor, but at lower levels than in B cells. IL-21 wasexpressed almost exclusively in stimulated CD4+ T cells (and not by CD8+T cells or CD19+ B cells). Although stimulation with PMA+Tonomycininduced a good IL-21 signal in this assay, a significantly higher signalwas obtained from CD4+ T cells stimulated with anti-CD3 mAb or acombination of anti-CD3 and anti-CD28 mAbs, conditions that better mimican antigen encounter in vivo.

Example 10

IL-21-dependent Proliferation of B-cell Cells Sstimulated Anti-CD40 orAnti-IgM

A. Purification of Human B Cells

A vial containing 1×10⁸ frozen, apheresed human peripheral bloodmononuclear cells (PBMCs) was quickly thawed in a 37° C. water bath andresuspended in 25 ml B cell medium (RPMI Medium 1640 (JRH Biosciences.Lenexa, Kans.), 10% Heat inactivated fetal bovine serum, 5% L-glutamine,5% Pen/Strep) (Gibco BRL)) in a 50 ml tube (Falcon VWR, Seattle, Wash.).Cells were tested for viability using Trypan Blue (Gibco BRL). Tenmilliliters of Ficoll/Hypaque Plus (Pharmacia LKB Biotechnology Inc.,Piscataway, N.J.) was layered under the cell suspension and spun for 30minutes at 1800 rpm and allowed to stop with the brake off. Theinterface was then removed and transferred to a fresh 50 ml Falcon tube,brought up to a final volume of 40 ml with PBS and spun for 10 minutesat 1200 rpm with the brake on. The viability of the isolated cells wasagain tested using Trypan Blue. Alternately fresh drawn human blood wasdiluted 1:1 with PBS (Gibco BRL) and layered over Ficoll/Hypaque Plus(Pharmacia), spun and washed as above. Cells isolated from either freshor frozen sources gave equivalent results.

B cells were purified from the Ficoll floated peripheral blood cells ofnormal human donors (above) with anti-CD19 magnetic beads (MiltenyiBiotec, Auburn, Calif.) following the manufacturer's instructions. Thepurity of the resulting preparations was monitored by flow cytometricanalysis with anti-CD22 FITC Ab (Pharmingen, SanDiego, Calif.). B cellpreparations were typically >90% pure.

B. Purification of Murine B Cells

A suspension of murine splenocytes was prepared by teasing adult C57B1/6mouse (Charles River Laboratories, Wilmington, Mass.) spleens apart withbent needles in B cell medium. RBCs were removed by hypotonic lysis.CD43 positive cells were removed with CD43 magnetic beads (MiltenyiBiotec) following the manufacturer's instructions. The purity of theresulting preparations was monitored by flow cytometric analysis withanti-CD45R FITC Ab (Pharmingen). B cell preparations were typically >90%pure.

C. Proliferation of anti-CD40-stimulated B-Cells in the Presence ofHuman or Murine IL-21

The B cells from either the human or mouse source were resuspended at afinal concentration of 1×10⁶ cells/ml in B cell medium and plated at 100Pl/well in a 96 well U bottom plate (Falcon, VWR) containing variousstimulation conditions to bring the final volume to 200 μl/well. Foranti-CD40 stimulation human cultures were supplemented with 1 μg/mlanti-human CD40 (Genzyme, Cambridge, Mass.) and mouse cultures weresupplemented with 1 μg/ml anti-murine CD40 (Serotec, UK). Human ormurine IL-21 was added at dilutions ranging from 1 μg/ml-100 ng/ml. Thespecificity of the effect of IL-21 was confirmed by inhibition of IL-21with 25 mg/ml soluble human zalphal ICEE (Example 10A). All treatmentswere performed in triplicate. The cells were then incubated at 37° C. ina humidified incubator for 120 hours (human) or 72 hours (mouse).Sixteen hours prior to harvesting, 1 μCi ³H-thymidine (Amersham,Piscataway, N.J.) was added to all wells to assess whether the B-cellshad proliferated. The cells were harvested into a 96 well filter plate(UniFilter GF/C, Packard, Meriden, Conn.) using a cell harvester(Packard) and collected according to manufacturer's instructions. Theplates were dried at 55° C. for 20-30 minutes and the bottom of thewells were sealed with an opaque plate sealer. To each well was added0.25 ml of scintillation fluid (Microscint-O, Packard) and the plate wasread using a TopCount Microplate Scintillation Counter (Packard).

Incubation with IL-21 at concentrations of 3 ng/ml or more enhanced theproliferation induced by soluble anti-CD40 in a dose dependent manner inboth murine and human B cells by as much as 30 fold. The murine andhuman B cells responded equally as well to their respective IL-21. Inboth species, the stimulation was specific to IL-21, as it was reversedby the presence of soluble IL-21 receptor in the culture.

D. Proliferation of Anti-IgM-stimulated B-Cells in the Presence of Humanor Murine IL-21

The B cells from either human or mouse source as described above(Example 10A and Example 10B) were plated as described above (Example10C). For anti-IgM stimulation of human cells the plates were pre-coatedovernight with 10 mg/ml F(ab′)₂ anti-human IgM Abs (Southern BiotechAssociates, Birmingham, Ala.) and washed with sterile media just priorto use. The cultures were supplemented with 0-10 ng/ml hu rIL-4 (R&DSystems, Minneapolis, Minn.). For anti-IgM stimulation of murine cellssoluble anti-IgM (Biosource, Camarillo, Calif.) was added to thecultures at 10 mg/ml. To each of the preceding anti-IgM/IL-4 conditions,human or murine IL-21 was added at dilutions ranging from 1 μg/ml-100ng/ml as described above. The specificity of the effect of IL-21 wasconfirmed by inhibition with soluble human zalpha11 receptor asdescribed above (Example 10C). All treatments were performed intriplicate. The cells were incubated, labeled with ³H-thymidine,harvested, and analyzed as described in Example 10C.

Incubation with IL-21 at concentrations of 0.3 ng/ml or more inhibitedthe proliferation induced by insoluble anti-IgM (mouse) or anti-IgM andIL-4 (human) in a dose-dependent manner. This inhibition was specific toIL-21 as it was reversed by the presence of soluble IL-21 receptor inthe culture.

Example 11

Human IL-21 Effect on B-cells and IL-21 Toxic Saporin Fusion

The effects of human IL-21 were tested on the following human B-celllines: and human Burkitt's lymphoma cell lines Raji (ATCC No.CCL-86),and Ramos (ATCC No. CRL-1596); human EBV B-cell lymphoma cell line RPMI1788 (ATCC No. CRL-156); human myeloma/plasmacytoma cell line IM-9 (ATCCNo. CRL159); and human EBV transformed B-cell line DAKIKI (ATCC No.TIB-206), and HS Sultan cells (ATCC No. CRL-1484 ). Following about 2-5days treatment with IL-21, changes in surface marker expression werefound in IM-9, Raji, Ramos, and RPMI1788 cell lines, showing that thesecells can respond to IL-21. Human B-cell lines treated with IL-21 grewmuch more slowly than untreated cells when re-plated in cell culturedishes. These cells also had an increased expression of FAS ligand, asassessed by flow cytometry (Example 11D and Example 11E), and moderatelyincreased sensitivity to an activating FAS antibody (Example 11A). Thisresults indicate that IL-21 could control some types of B-cell neoplasmsby inducing them to differentiate to a less proliferative and or moreFAS ligand sensitive state. Moreover, zalpha11 receptor is expressed onthe surface of several of these cell lines (U.S. Pat. No. 6,307,024).Thus, IL-21 and the human IL-21-saporin immunotoxin conjugate (Example11B, below), or other IL-21-toxin fusion could be therapeutically usedin B-cell leukemias and lymphomas.

A. The Effect of Human IL-21 on B-cell Lines.

IM-9 cells were seeded at about 50,000 cells per ml±50 μg/ml purifiedhuman IL-21 (U.S. Pat. No. 6,307,024). After 3 days growth the cellswere harvested, washed and counted then re-plated at about 2500 cells/mlin 96 well plates in to wells with 0, 0.033, 0.1 or 0.33 μg/ml anti-FASantibody (R&D Systems, Minneapolis). After 2 days an Alamar bluefluorescence assay was performed (U.S. Pat. No. 6,307,024) to assessproliferation of the cells.

IL-21-treated IM-9 cells grew to only 27% the density of the untreatedcells in the absence of anti-FAS antibody. In the presence of 0.33 μg/mlanti-FAS antibody, the IL-21-treated cells were inhibited an additional52% while the untreated cells were inhibited by only 30%. The overallinhibition of cell growth with both IL-21 and 0.33 μg/ml anti-FASantibody treatment was 86%.

When the IM-9 cells were pretreated for three days with or without IL-21and then re-plated at 100 cells per well and grown with or withoutanti-FAS antibody for 6 days, the growth of untreated cells assessed byAlamar Blue assay (U.S. Pat. No. 6,307,024) was inhibited only 25% byanti-FAS antibody while the growth of IL-21-treated cells was inhibited95% relative to the growth of untreated cells in zero anti-FAS antibody.

B. The Effect of Human IL-21-saporin Immunotoxin on B-cell Lines.

The human IL-21-saporin immunotoxin conjugate (zalpha11L-sap)construction and purification is described in Example 12. The humanzalphal IL-sap was far more potent than the saporin alone in inhibitingcell growth. When the treated cell are re-plated after a three or fourday treatment the human zalpha11L-sap treated cells grew very poorly.

IM-9, Ramos and K562 (ATCC No. CCL-243) cells were seeded at about 2500cells/well in 96 well plates with zero to 250 ng/ml human zalpha11L-sapconjugate or 0-250 ng/ml saporin (Stirpe et al., Biotechnology10,:405-412, 1992) only as a control. The plates were incubated 4 daysthen an Alamar Blue proliferation assay was performed (U.S. Pat. No.6,307,024). At the maximal concentration of human zalpha11-sapconjugate, the growth of IM-9 cells and RAMOS cells was inhibited by 79%and 65% respectively. K562 cells which are low/negative by flow forexpression of the IL-21 receptor were not affected by the zalpha11-sap,thus showing the specificity of the conjugate's effect.

IM-9 cells were seeded a 50,000 cells/ml into 6 well plates at zero and50 ng/ml human zalpha11L-sap conjugate. After 3 days the cells wereharvested and counted then re-plated from 100 to 0.8 cells per well in 2fold serial dilutions, and 12 wells per cell dilution without the humanIL-21-saporin immunotoxin. After 6 days the number of wells with growthat each cell dilution was scored according to the results of an Alamarblue proliferation assay (U.S. Pat. No. 6,307,024).

When cell number was assessed, by Alamar blue assay (U.S. Pat. No.6,307,024), after 6 days of growth control cells seeded at about 12.5and 6.25 cells per well had equivalent growth to zalpha11-sap treatedcells seeded at 100 and 50 cells/well respectively. Thus, the growth ofthe surviving treated IM-9 cells was markedly impaired even after theremoval, by re-plating, of the zalpha11-sap immunotoxin.

The limited tissue distribution of the human IL-21 receptor (U.S. Pat.No. 6,307,024 and WIPO Publication No.s WO 0/17235 and WO 01/7717), andthe specificity of action of the zalpha11-sap to receptor-expressingcell lines suggest that this conjugate may be tolerated in vivo.

C. The Effect of Human IL-21-saporin Immunotoxin on B-cell LineViability.

HS Sultan cells (ATCC No. CRL-1484 ) were seeded at about 40,000 cellsper ml into 12 well plates and grown for five days with either no addedcytokines or 40 ng/ml purified human IL-21 (U.S. Pat. No. 6,307,024) or25 ng/ml human zalpha11L-sap conjugate (Example 12, below) or with 20ng/ml IFN-alpha (RDI) or IL-21 and IFN-alpha. IL-21 inhibited theoutgrowth of Hs Sultan cells by 63%. IFN-alpha inhibited the growth by38%. IL-21 plus IFN-alpha inhibited growth 78%, indicating that thegrowth inhibitory effects of human IL-21 and IFN-alpha may be additive.The human zalpha11L-sap inhibited growth of the HS Sultans by 92%.

The results above support the possible use of IL-21 or humanzalpha11L-sap in the treatment of malignancies or other diseases thatexpress the zalpha11 receptor, particularly those of B-cell origin. Thecombination of IL-21 with IFN-alpha is specifically suggested by theiradditive effect in the inhibition of HS Sultan cells. Some other typesof lymphoid malignancies and diseases may also express the IL-21receptor, as activated T-cells also express the receptor mRNA (U.S. Pat.No. 6,307,024 and WIPO Publication No.s WO 0/17235 and WO 01/7717) andsome of these diseases may also be responsive to IL-21 of IL-21-toxicfusion therapy.

D. FAS (CD95) Expression on Human B-cell Lines is Increased by HumanIL-21 Stimulation

Human B-cell lines HS Sultan (ATCC No. CRL-1484), IM-9 (ATCC No.CRL159), RPMI 8226 (ATCC No. CCL-155), RAMOS (ATCC No. CRL-1596), DAKIKI(ATCC No. TIB-206), and RPMI 1788 (ATCC No. CRL-156), were all treatedwith or without purified 10 to 50 ng/ml human IL-21 (U.S. Pat. No.6,307,024) for 2 to 8 days. The cells were then stained with anti-CD95PE-conjugated antibody (PharMingen, San Diego, Calif.), permanufacturer's protocol, and analyzed on a FACScalibur (BectonDickinson, San Jose, Calif.). In all cell lines, anti-CD95 (FAS orAPO-1) staining was increased, in some cases more than two fold, upontreatment with human IL-21.

E. FAS (CD95) Expression on Primary Mouse Spleen B-cells is Increased byHuman IL-21 Stimulation

Primary mouse splenocytes were obtained by chopping up spleens from 8 to12 week old C57/BL6 mice. Erythrocytes were lysed by treating thepreparation for 5 seconds with water then put through a 70 micron sieve.The remaining splenocytes were washed and plated in RPMI (JRHBioscience) plus 10% HIA-FBS (Hyclone, Logan, Utah). IL-2 (R & DSystems) with or without human IL-21, as described above. They were thenincubated at 37° C., in 5% CO₂ for 5 days. The splenocytes wereharvested and stained with anti-CD95 PE conjugated antibody (PharMingen)and anti-CD19 FITC conjugated antibody (PharMingen) per manufacturer'sprotocol. The cells were analyzed by flow cytometry on a FACScalibur(Becton Dickinson). Upon gating on the CD19+ mouse B-cells, it was foundthat anti-CD95 staining was increased on B-cells treated with IL-2 plushuman IL-21 compared to those in IL-2 alone. The anti-CD95 staining was37 relative fluorescent units (RFU) on the B-cells in IL-2 alone and 55RFU on the B-cells cultured in IL-2 and human IL-21.

Example 12

Construction and Purification of IL-21 Toxic Fusion

Under a supply contract, 10 mg human IL-21 (U.S. Pat. No. 6,307,024) wassent to Advanced Targeting Systems (ATS, SanDiego, Calif.) forconjugation to the plant toxin saporin (Stirpe et al., Biotechnology10,:405-412, 1992). ZymoGenetics received from ATS 1.3 mg of a proteinconjugate comprised of 1.1 molecules saporin per molecule of humanIL-21, formulated at a concentration of 1.14 mg/ml in 20 nM Sodiumphosphate, 300 nM sodium cloride, pH 7.2.

Example 13

IL-21 Toxic Fusion In Vivo

A. Testing IL-21-saeorin Conjugate in Mice

IL-21-saporin conjugate (Example 11) was administered to C57BL6 mice(female, 12 weeks of age, purchased from Taconic) at two differentdosages: 0.5 and 0.05 mg/kg. Injections were given i.v. in vehicleconsisting of 0.1% BSA (ICN, Costa Mesa, Calif.). Three injections weregiven over a period of one week (day 0, 2, and 7). Blood samples weretaken from the mice on day 0 (pre-injection) and on days 2 and 8(post-injection). Blood was collected into heparinized tubes (BectinDickenson, Franklin Lakes, N.J.), and cell counts were determined usingan automated hematology analyzer (Abbot Cell-Dyn modelNo. CD-3500CS,Abbot Park, Ill.). Animals were euthanized and necropsied on day 8following blood collection. Spleen, thymus, liver, kidney and bonemarrow were collected for histopathology. Spleen and thymus wereweighed, and and additional blood sample was collected in serumseparator tubes. Serum was sent to Pheonix Central Labs, Everett, Wash.,for testing in a standard chemistry panel. Samples were also collectedfor flow cytometric analysis as described herein.

Circulating blood cell counts and serum chemistry measurements did notdiffer significantly between IL-21 conjugate treated mice and micetreated with an equivalent dose of unconjugated toxin (saporin).Histological analysis of tissues in IL-21-saporin treated mice showed nosignificant changes relative to mice treated with an equivalent dose ofunconjugated toxin. These results indicated that the saporin conjugatewas not toxic in vivo.

B. Testing IL-21 Toxic Saporin Fusion on B-cell Derived Tumors In Vivo

The effects of human IL-21 and the human IL-21 toxic saporin fusion(Example 12) on human tumor cells are tested in vivo using a mouse tumorxenograft model described herein. The xenograft models are initiallytested using cell lines selected on the basis of in vitro experiments,such as those described in Example 11. These cell lines include, but arenot limited to: human Burkitt's lymphoma cell lines Raji (ATCCNo.CCL-86), and Ramos (ATCC No. CRL-1596); human cell line RPMI 1788(ATCC No. CRL-156); human myeloma/plasmacytoma cell line IM-9 (ATCC No.CRL159); human cell line DAKIKI (ATCC No. TIB-206), and HS Sultan cells(ATCC No. CRL-1484). Cells derived directly from human tumors can alsobe used in this type of model. In this way, screening of patient samplesfor sensitivity to treatment with IL-21 or with a IL-21 toxic saporinfusion can be used to select optimal indications for use of zalpha11 inanti-cancer therapy.

After selection of the appropriate zenograft in vivo model, describedabove, IL-21-induced activity of natural killer cells and/or IL-21effects on B-cell derived tumors are assessed in vivo. Human IL-21 istested for its ability to generate cytotoxic effector cells (e.g. NKcells) with activity against B-cell derived tumors using mouse tumorxenograft models described herein. Moreover, direct affects of humanIL-21 on tumors can be assessed. The xenograft models to be carried outare selected as described above. A protocol using IL-21 stimulated humancells is developed and tested for efficacy in depleting tumor cells andpromoting survival in mice innoculated with cell lines or primarytumors.

Example 14

Preliminary Evaluation of the Aqueous Stability of Human IL-21

Preliminary studies were conducted to evaluate the aqueous stabilitycharacteristics of human IL-21 in support of bioprocessing, formulation,and in vivo administration. The objectives were to: 1) verify thestability and recovery from Alzet Minipumps & general storage andhandling, 2) determine the stability-indicating nature of severalanalytical methods including ca tion-exchange HPLC (CX-HPLC),reverse-phase HPLC (RP-HPLC), size exclusion HPLC (SEC-HPLC), & bioassay(BaF3/zalpha11R proliferation (e.g., U.S. Pat. No. 6,307,024), and 3)determine the stability-limiting degradation pathways and their kineticdependencies.

Aliquots of purified human IL-21 (U.S. Pat. No. 6,307,024) were preparedby dilution to 2 mg/mL in PBS (pH 7.4) and stored in low densitypolyethylene (LDPE) cryovials (Nalgene, 1.8 mL) at −80° C. (control), 5°C., 30° C., and 37° C. Samples were assayed intermittently over 29 daysby CX-, RP-, SEC-HPLC, and bioassay. Aliquots were also stored at −80°C. and subjected to freeze-thaw (f/t) cycling (−80° C./RT; 5× f/t, 10×f/t). Recovery of human IL-21 was determined relative to the −80° C.control (1 f/t) in all assays.

The remaining human IL-21 solution from the −80° C. control samples wererefrozen (−80° C.) after analysis. This aliquot (2 f/t) was used toevaluate the thermal and conformational stability of human IL-21 as afunction of pH using circular dichroism (CD). The 2 mg/mL solution wasdiluted to 100 μg/mL in PBS buffers ranging from pH 3.3-8.8. The far-UVCD spectra was monitored over the temperature range 5-90° C. in 5° C.intervals (n=3/pH). The CD spectropolarimeter used was a Jasco 715(Jasco, Easton, Md.). The thermal unfolding was monitored by changes inellipticity at 222 nm as a function of temperature. Estimates of the Tmwere estimated assuming a two-state unfolding model. The data was fit(sigmoidal) using SlideWrite Plus for Windows v4.1 (Advanced GraphicsSoftware; Encinitas, Calif.).

Recovery and stability from Alzet Minipumps (Model No. 1007D; ALZACorporation, Mountain View, Calif.) was assessed by filling pumps with100 μL of the 2 mg/mL human IL-21 solution, placing the pumps in 1.8 mLLDPE containing 1 mL of PBS (pH 7.4), and storing them at 37° C. Therelease/recovery of human IL-21 from the minipumps was assessed by CX-,RP-, and SEC-HPLC on days 2, 4, and 7. The activity was assessed bybioassay on day 7. The study was designed to evaluate the release from 3pumps per sampling time.

The chromatographic data suggested that the CX- & SEC-HPLC methods werestability-indicating, whereas the RP-HPLC method was not. At least 3additional peaks indicating apparent degradation products were observedby CX-HPLC. The SEC-HPLC method resolved an apparent human IL-21aggregate, eluting prior to human IL-21. However, no significantadditional peaks were observed eluting after the human IL-21 peak. Thissuggests that the degradation products observed by CX-HPLC most probablyresult from amino acid modifications such as deamidation, rather thanhydrolysis/proteolysis processes leading to clipped variants. A smalldegree of fronting/tailing was observed by RP-HPLC (relative to control)in samples which had been shown to have undergone significantdegradation by SEC- & CX-HPLC. However, apparent degradation productswere not resolved by RP-HPLC. The degradation observed by CX-HPLCincreased as a function of time-temperature, and followed apparentfirst-order kinetics. The % human IL-21 recovered by CX-HPLC after 29days at 37° C., 30° C., and 5° C. was 39%, 63%, and 98%, respectively.Aggregation also increased in a time-temperature dependent fashion. The% aggregate found in preparations stored for 29 days at 37° C., 30° C.,and 5° C. was 7.4, 3.4, and below detectable limits (BDL), respectively.No significant differences were observed by bioassay in any sample,suggesting the degradation products have equivalent activity to intacthuman IL-21. No degradation was observed by any assay in samplessubjected to up to 10 f/t cycles.

The release of human IL-21 from Alzet Minipumps was consistent with thetheoretical expected volume release. This suggests that significantsurface adsorption would not impair the delivery of human IL-21 usingthe Alzet Minipumps with a 2 mg/mL fill concentration. The degradationconsistent with that previously noted was observed. The % puritydetermined by CX-HPLC of human IL-21 released after 2, 4, and 7 days was96%, 90%, and 79%, repectively. It should be recognized that degradationalso occurs after human IL-21 is released into or diluted with releasemedium. Therefore, the % purity within the minipump may be somewhatdifferent than that determined to be in the release medium. Thebioactivity of each sample was consistent with the expected amount ofhuman IL-21 released from the minipumps.

The human IL-21 far-UV CD spectra, as expected, was consistent withinterleukins, such as IL-3 (J. Biochem., 23:352-360, 1991), IL-4(Biochemistry, 30:1259-1264, 1991), and IL-6 mutants (Biochemistry,35:11503-11511, 1996). Gross changes in the far-uv CD spectra as afunction of pH were not observed. Results showed that the pH of maximumthermalyconformational stability was pH 7.4. Analysis of the unfoldingcurves were based on a two-state unfolding mechanism to allow comparisonof the thermal/conformational stability as a function of pH/composition.However, one or more intermediates may exist during the unfoldingprocess since the cooperativity was relatively low, based on theshallowness of the unfolding curve. Although studies were notspecifically designed to determine whether human IL-21 refolds followingthermal unfolding to 90° C., preliminary data suggests that at leastpartial refolding occurs after the temperature of the sample is cooledback to 20° C.

These studies allow an analytical paradigm to be identified to evaluatethe purity and verify the stability of human IL-21. For instance,SEC-HPLC can be used to characterize the extent and rate of aggregationin aqueous solution. Likewise, CX-HPLC can be used to characterize theextent and rate of degradation of human IL-21 by mechanisms other thanaggregation. The bioassay can be used to verify activity of human IL-21and it's aqueous degradation products. For instance, the human IL-21variants generated in aqueous solution & resolved by CX-HPLC maythemselves be useful as therapeutic agents, since they have equivalentbioactivity. Also, the fact that human IL-21 degrades by severaldifferent processes (aggregation, amino acid modifications) suggests apreferred or unique formulation which minimizes the rate of eachdegradation process may be necessary for long-term stability of asolution product.

Identification of the nature of the aqueous degradation products anddetermination of their kinetic dependencies (pH, concentration,excipients) is underway. Human IL-21 stability in serum/plasma isdetermined to support the design and interpretation of in vivo studies.

Example 15

IL-21 Effect on B-cell Dderived Ttumors In Vivo

A. Infusion of IL-21 Using Mini-osmotic Pumps

Administration of IL-21 by constant infusion via mini-osmotic pumpsresulted in steady state serum concentrations proportional to theconcentration of the IL-21 contained in the pump. 0.22 ml of human IL-21(U.S. Pat. No. 6,307,024) contained in phosphate buffered saline (pH6.0) at a concentration of 2 mg/ml or 0.2 mg/ml was loaded under sterileconditions into Alzet mini-osmotic pumps (model 2004; Alza corporationPalo Alto, Calif.). Pumps were implanted subcutaneously in mice througha 1 cm incision in the dorsal skin, and the skin was closed with sterilewound closures. These pumps are designed to deliver their contents at arate of 0.25 μl per hour over a period of 28 days. This method ofadministration resulted in significant increase in survival in miceinjected with tumor cells (below).

B. IL-21 Effect on B-cell Derived Tumors In Vivo

The effects of human IL-21 (U.S. Pat. No. 6,307,024) were tested in vivousing a mouse tumor xenograft model described herein. The xenograftmodels tested were human lymphoblastoid cell line IM-9 (ATCC No.CRL159). C.B-17 SCID mice (female C.B-17/IcrHsd-scid; Harlan,Indianapolis, Indiana) were divided into 4 groups. On day 0, IM-9 cells(ATCC No. CRL159) were harvested from culture and injectedintravenously, via the tail vein, to all mice (about 1,000,000 cells permouse). On day 1, mini-osmotic pumps containing test article or controlarticle were implanted subcutaneously in the mice. Mice in groups 1-3(n=9 per group) were treated with increasing concentrations of IL-21:group 1 contained 2.0 mg/mL of human IL-21 and delivered 12 μg per day;group 2 contained 0.20 mg/mL of human IL-21 and delivered 1.2 μg perday; group 3 contained 0.02 mg/mL of human IL-21 and delivered 0.12 μgper day. Mice in group 4 (n=9) were a control and were treated withvehicle (PBS pH 6.0).

Mice treated with either 12 μg/day or 1.2 μg/day IL-21 infusion hadincreased survival compared to vehicle treated mice (p<0.0001 andp<0.005 for 12 μg/day or 1.2 μg/day vs. vehicle, respectively, using logrank tests of the survival function). Mice in the 0.12 μg/day dose grouphad survival no different than the mice in the vehicle treated group.These results showed that IL-21 significantly reduced the effects of theB-cell tumor cells in vivo, significantly resulting in increasedsurvival.

Example 16

In Vivo Anti-tumor Effects of IL-21 in B16-F10 Melanoma and EG.7 ThymomaModels

A. Murine IL-21 Effect on B16-F10 Melanoma Metastasis Growth In Vivo

Mice (female, C57B16, 9 weeks old; Charles River Labs, Kingston, N.Y.)were divided into three groups. On day 0, B16-F10 melanoma cells (ATCCNo. CRL-6475) were harvested from culture and injected intravenously,via the tail vein, to all mice (about 100,000 cells per mouse). Micewere then treated with the test article or associated vehicle byintraperitoneal injection of 0.1 ml of the indicated solution. Mice inthe first group (n=24) were treated with vehicle (PBS pH 6.0), which wasinjected on day 0, 2, 4, 6, and 8. Mice in the second group (n=24) weretreated with murine IL-21 (U.S. Pat. No. 6,307,024), which was injectedat a dose of 75 μg on day 0, 2, 4, 6, and 8. Mice in the third group(n=12) were treated with murine IL-21, which was injected at a dose of75 μg daily from day 0 through day 9. All of the mice were sacrificed onday 18, and lungs were collected for quantitation of tumor. Foci oftumor growth greater than 0.5 mm in diameter were counted on allsurfaces of each lung lobe. In both groups of mice treated with murineIL-21, the average number of tumor foci present on lungs wassignificantly reduced, compared to mice treated with vehicle. Micetreated more frequently (i.e. daily) had fewer tumor foci than micetreated on alternate days, although this was not a statisticallysignificant finding between these two groups.

These results indicated that treatment with murine IL-21 either slowedthe growth of the B16 melanoma tumors or enhanced the ability of theimmune system to destroy the tumor cells. The effects of the treatmenton tumor cells were likely mediated through cells of the immune system(i.e. lymphocytes, NK cells), which do possess receptors for IL-21, suchas, for example, IL-21 receptor and zalpha11/IL-2Rγ (WIPO PublicationNo.s WO 0/17235 and WO 01/7717) and are known to be associated withanti-tumor activity.

B. Murine IL-21 Effect on EG.7 Thymoma Growth In Vivo

Mice (female, C57B16, 9 weeks old; Charles River Labs, Kingston, N.Y.)were divided into three groups. On day 0, EG.7 cells (ATCC No. CRL-2113)were harvested from culture and 1,000,000 cells were injectedintraperitoneal in all mice. Mice were then treated with the testarticle or associated vehicle by intraperitoneal injection of 0.1 mL ofthe indicated solution. Mice in the first group (n=6) were treated withvehicle (PBS pH 6.0), which was injected on day 0, 2, 4, and 6. Mice inthe second group (n=6) were treated with murine IL-21 (U.S. Pat. No.6,307,024), which was injected at a dose of 10 μg on day 0, 2, 4, and 6.Mice in the third group (n=6) were treated with murine IL-21, which wasinjected at a dose of 75 μg on day 0, 2, 4, and 6. In both groups ofmice treated with murine IL-21, time of survival was significantlyincreased, compared to mice treated with vehicle. The group treated with75 μg doses of IL-21 had significantly greater survival than the grouptreated with 10 μg doses, and 33% (2/6 mice) of this group survivedlonger than 70 days. An additional portion of this study tested theeffect of the same dosages carried out through day 12. The results werevery similar to the shorter dosing schedule, with both doses havingsignificantly increased survival over vehicle treatment, and the highestdose gave the best response (50% survival past 70 days).

In some experiments about 4,000,000 OT-1 T cells were injectedintraperitoneally in the mice on the day prior to day 0. The mice werethen treated with IL-21 or vehicle as above. The presence of the OT-1 Tcells had no effect on the survival time of the vehicle treated mice. Inmice treated with IL-21 the presence of the OT-1 T cells enhancedsurvival time compared to the mice treated with IL-21 alone.

These results indicated that treatment with murine IL-21 either slowedthe growth of the EG.7 tumors or enhanced the ability of the immunesystem to destroy the tumor cells. The increase in survival conferred bythe OT-1 T cells in the presence of IL-21 treatment suggests that IL-21is activating effector cells of the immune system.

Example 17

IL-21 Effects on Serum Cytokines and Vascular Leak

A. Analysis of IL-21 on Serum Cytokines

IL-2 therapy is effective in the treatment of certain cancers. However,the use of IL-2 as a therapeutic agent has been limited by its toxiceffects, namely vascular leak syndrome (VLS). IL-2 induced VLS ischaracterized by infiltration of lymphocytes, monocytes and neutrophilsinto the lung causing endothelial damage in the lung eventually leadingto vascular leak (reviewed in Lentsch A B et al, Cancer Immunol.Immunother., 47:243, 1999). VLS in mice can be induced withadministration of repeated high doses of IL-2 and measuring vascularleak by Evan's Blue uptake by the lung. Other parameters that have beenshown to be characteristic of VLS in mice include increased serum levelsof TNFα and IFNγ (Anderson J A et al, J. Clin. Invest. 97:1952, 1996) aswell as increased numbers of activated T, NK and monocytes in variousorgans. Blocking of TNFα with a soluble TNFR-Fc molecule inhibited lunginfiltration by lymphocytes and therefore lung injury (Dubinett S M etal, Cell. Immunol. 157:170, 1994). The aim was to compare the ability ofIL-2 and IL-21 to induce VLS in mice and to measure the differentparameters indicative of VLS (Evan's Blue uptake, serum cytokineanalysis, spleen cellular phenotype).

Mice (female, C57B16, 11 week old; Charles River Labs, Kingston, N.Y.)were divided into five groups. All groups contained 10 mice per group.Groups are as follows: Group I or Vehicle group received PhosphateBuffered Saline (PBS); Group II and III received IL-2 0.6 or 1.8 millionIU/injection respectively; Group IV and V received mouse IL-21 (U.S.Pat. No. 6,307,024) or 100 μg/injection respectively. The studyconsisted of 4 days, body weight was measured daily and animals received7 intraperitoneal injection of test substance over the 4-day period.Animals received two daily injections on day 1-3 and on the fourth dayreceived a single morning injection. Two hours post final injectionanimals received a tail vein injection of 1% Evan's blue (0.2 ml). Twohours post Evan's blue injection mice where anesthetized with Isofluraneand blood was drawn was serum cytokine analysis. Following blood drawanimals where transcardial perfused with heparinized saline (25 U hep/mlsaline). Following perfusion spleen was removed and weighed, liver andlung where removed and placed into 10 mls of formamide for 24 hrincubation at room temperature. Following 24 hr incubation vascularleakage was quantitated by Evan's blue extravasation via measurement ofthe absorbance of the supernatant at 650 nm using a spectrophotometer.

Mice were bled and serum separated using a standard serum separatortube. 25 μl of sera from each animal was used in a Becton Dickenson (BD)Cytokine Bead Array (Mouse Th1/Th2 CBA Kit) assay. The assay was done asper the manufacturer's protocol. Briefly, 25 μl of serum was incubatedwith 25 μl bead mix (IL-2, IL-4, IL-5, TNFα and IFNγ) and 25 μlPE-detection reagent for two hours at room temperature in the dark. Aset of cytokine standards at dilutions ranging from 0-5000 μg/ml wasalso set up with beads as per the manufacturer's instructions. Theincubated beads were washed once in wash buffer and data acquired usinga BD FACScan as per instructions outlined in the Kit. The data wasanalyzed using the BD Cytometric Bead Array Software (BD Biosciences,San Diego, Calif.).

Serum cytokine analysis using the CBA cytokine kit (Becton Dickenson,San Diego, Calif.) showed no increases in levels of IL-2, IL-4, IL-5,IFNγ or TNFα in the PBS control treated groups. There was a dosedependent increase in the levels of IL-5, IFNγ and TNFα in sera fromIL-2 treated mice. There was no increase in the levels of the 5 measuredcytokines in the serum of mice treated with IL-21. The cytokine levelsin the highest dose of IL-21 mirrored that of the PBS treated animals.This shows that unlike IL-2 treatment that leads to increase in serumlevels of the inflammatory cytokines IL-5, TNFα and IFNγ, treatment withIL-21 does not have any effect on these inflammatory cytokines.

The results from a representative experiment is given in Table 6. Allconcentrations are expressed in μg/ml were an average of 4animals/group.

As shown in Table 5 above, treatment of mice with IL-2 resulted in adramatic increase in serum inflammatory cytokines, namely IL-5, IFNγ andTNFα. Treatment of mice with IL-21 did not show any increase in cytokinelevels above PBS treated mice. These results show that even at thehighest doses, IL-21 does not upregulate inflammatory cytokines and it'seffect on cells in vivo is different from IL-2.

Treatment of mice with repeated high dose IL-2 resulted in increasedserum levels of IL-5, IFNγ and TNFα. These pro-inflammatory cytokineshave been shown to play a role in VLS associated with IL-2 toxicity.Blocking TNFa resulted in decreased lymphocyte infiltration into thelungs and decreased lung injury associated with IL-2 toxicity (DubinettS M et al, 1994, Cell. Immunol. 157:170). IL-21 treatment did not haveany effects on serum IL-5, TNFα or IFNγ levels. This suggests that IL-21acts different from IL-2 in vivo and that TABLE 6 TNFα IFNγ IL5 IL4 IL2PBS 2.4 0.0 2.9 2.2 1.3 IL2 0.6 millU 22.9 21.6 1095.0 2.5 2.0 IL2 1.8millU 69.1 185.1 1132.9 2.0 1.9 IL2 3.6 millU 78.9 195.6 651.3 1.8 2.1IL-21 3 μg 2.1 1.6 2.7 1.7 1.6 IL-21 100 μg 3.1 0.0 4.0 0.0 1.1 IL-21200 μg 7.3 1.9 4.0 2.6 1.9

the lack of pro-inflammatory cytokines in sera of IL-21 treated micemight indicate lesser toxicity of IL-21 compared to IL-2.

B. Analysis of IL-21 on Vascular Leak—Immunophenotyping of Splenic Cells

IL-2 induced vascular leak syndrome (VLS) involves organ damage thatoccurs at the level of postcapillary endothelium. However, this damageoccurs secondary to two distinct pathological processes: the developmentof VLS, and transendothelial migration of lymphocytes. Acute organinjury is mediated by infiltrating neutrophils while chronic organinjury is mediated by infiltration monocytes and lymphocytes (reviewedin Lentsch A B et al, supra.). In mice, depletion of cells with surfacephenotypes characteristic of LAK or NK cells ameliorates organ damage(Anderson T D et al, Lab. Invest. 59:598, 1988; Gately, M K et al. J.Immunol., 141:189, 1988). Increased numbers of NK cells and monocytes istherefore a marker for IL-2 mediated cellular effects of VLS. Inaddition, IL-2 directly upregulates the expression of adhesion molecules(i.e LFA-1, VLA-4 and ICAM-1) on lymphocytes and monocytes (Anderson J Aet al, supra.). This increase is thought to enable cells to bindactivated endothelial cells and help in transmigration of cells to thetissue. Increased expression of these molecules is considered anothermarker of IL-2 induced cellular activation during VLS. The aim of thisstudy was to study splenic cells from IL-2 and IL-21 treated mice undera VLS protocol and compare the effects of the two cytokines to mediatecellular effects associated with VLS.

Groups of age and sex matched C57BL/6 mice treated and described above(Example 17A) were analysed. On d4, mice were sacrificed and phenotypeof splenic cell populations studied by standard flow cytometry. Splenicweight and cellularity were dramatically increased in IL-2 treated micecompared to PBS treated mice. IL-21 treated mice had a slight increasein splenic weights (at the higher doses) but no significant increase insplenic cellularity compared to the PBS treated groups. Cell populationanalysis showed a significant increase in the percentage and numbers ofNK, NKT and monocytes in IL-2 treated mice but not in the IL-21 treatedmice. Furthermore, there was a dose dependent dramatic increase in LFA-1expressing cells in the IL-2 treated groups compared to PBS controls.IL-21 treatment had no effect on LFA-1 expression on splenic cells.

Spleens were isolated from mice from the various groups. Red blood cellswere lysed by incubating cells for 4 minutes in ACK lysis buffer (0.15MNH4Cl, 1 mM KHCO3, 0.1mM EDTA) followed by neutralization in RPMI-10media (RPMI with 10% FBS). The expression of cell surface markers wasanalyzed by standard three color flow cytometry. All antibodies wereobtained from BD Pharmingen (San Diego, Calif.).Fluorescin-isothiocyanate (FITC) conjugated CD11a (LFA-1), CD49d (VLA-4,a chain), Gr-I FITC, phycoerythrin (PE) conjugated CD4, NK1.1, CD11b andCyC-conjugated CD8, CD3 and B220 were used to stain cells. 1-3×10⁶ cellswere used for individual stains. Non-specific binding was blocked byincubating cells in blocking buffer (PBS, 10% FBS, 20 ug/ml 2.4G2).After blocking, cells were incubated with primary antibodies for 20minutes. Unless specified otherwise, all mAbs were used at 1 ug/stain ina volume of 100 ul. Cells were washed once in 1× PBS and resuspended inPBS before being acquired using the FACScan or FACSCalibur instruments(BD Biosciences, San Diego, Calif.). Data was analyzed using theCellquest Software (BD Biosciences).

IL-2 treated mice had significantly increased spleen weights compared toPBS treated groups (Table 7, below) IL-21 treated mice had significantincrease in spleen weight over controls. However, the increases in IL-21treated groups were significantly less than in the IL-2 treated groups(p=0.0002). The increase in spleen weights in both groups was dosedependent. Table 7 below shows the treatment groups; the average splenicweights were shown in mg, and n=4. TABLE 7 Total spleen weight (mg)Stdev p value (vs PBS) PBS 63.5 9.7 0 IL-2 (0.6) 177.5 17.8 <0.0001 IL-2(1.8) 204.25 10 <0.0001 IL-2 (3.6) 231.2 9.6 <0.0001 IL-21 (33) 92.8 60.0022 IL-21 (100) 117.6 19.3 0.0024 IL-21 (200) 125.85 33 0.0111

Average Splenic cellularity data is shown in Table 8, below (n=4).Higher dose IL-2 treatment increased splenic cellularity significantlyover control PBS treated groups. IL-21 treated groups did not showsignificant increase in splenic cellularity compared to PBS groups.TABLE 8 Total cells (×10⁶) Stdev p value (vs PBS) PBS 48 16.4 o IL-2(0.6) 57.1 11.8 0.4014 IL-2 (1.8) 100.4 21.6 <.0083 IL-2 (3.6) 101.84.25 <.0007 IL-21 (33) 58.8 13.5 0.3463 IL-21 (100) 48 7.83 0.9769 IL-21(200) 53.8 22.5 0.6917

IL-2 induced VLS is characterized by increased numbers of NK cells,monocytes and cells expressing the adhesion marker LFA-1 (reviewed inLentsch AB et al, supra.). The above data using IL-2 reproducespublished reports on the increase of NK cells, monocytes and LFA-1+cells. IL-2 treated mice show all signs of VLS compared to controls. Incontrast, IL-21 treated mice, although having an increased uptake ofEvan's Blue, show no increase in serum pro-inflammatory cytokines, orincrease in LFA-1+ cells or NK cells. Furthermore, although IL-21treated mice do show increased numbers of monocytes, the increase isless than what is seen with IL-2 treated animals, suggesting that IL-2mediated effects are more severe than IL-21 mediated effects. Takingtogether the splenic cellularity data and the serum cytokine data, IL-21does not induce a comparable inflammatory response as IL-2. Allparameters analyzed would indicate that IL-21 induces minor if anyinflammatory response when administered in a VLS protocol in similardoses to IL-2 (weight/weight).

In addition, as shown in Table 9 and Table 10, below, flow cytometryanalysis of spleen cells from mice revealed that IL-2 treated mice had adose dependent increase in the % and numbers of splenic NK/T cells(NK1.1+CD3+), NK cells (NK1.1+CD3−), macrophages (CD11b+) and LFA-1+cells (TABLE III and IV). IL-21 treated mice had no increase in NK/Tcells, NK cells or LFA-1+ cells. There was an increase in the % andnumbers of macrophages and granulocytes (data not shown) in IL-21treated group compared to the control PBS treated groups. This increasewas similar or less than the increase in IL-2 treated mice. TABLE 9Average % of lineage cells in spleen (n = 4) % % LFA- % NK/T % NKmacrophages % B % CD4 T % CD8 T 1+ PBS 0.7225 3.1925 6.625 50.025 21.97514.275 11.1775 IL-2 (0.6) 4.34 9.3375 12.525 43.9 17.65 10.15 29.26 IL-2(1.8) 3.2 13.9 14.525 43.75 15.55 11.55 34.825 IL-2 (3.6) 3.075 14.311.875 42.8 14.875 17.325 44.05 IL-21 (33) 0.615 2.9875 7.825 53.6517.925 10.95 8.4375 IL-21 (100) 0.63 2.76 11.375 48.325 17.825 11.27513.35 IL-21 (200) 1.025 3.4325 16.175 45.125 17.225 12.15 15.7

TABLE 10 Splenic cell numbers (×10⁶ cells, n = 4) NKT NK CD11b B220 Cd4Cd8 LFA-1 Gr-1 PBS 0.33 1.54 3.19 24.34 10.19 6.58 5.41 1.11 IL-2 (0.6)2.34 5.33 7.04 25.23 10.16 5.77 16.60 2.82 IL-2 (1.8) 3.08 14.29 14.1643.63 15.57 11.62 35.11 7.65 IL-2 (3.6) 3.15 14.59 12.08 43.53 15.1317.64 44.83 4.76 IL-21 (33) 0.37 1.77 4.67 31.50 10.47 6.32 5.05 1.05IL-21 0.30 1.33 5.35 23.16 8.55 5.40 6.35 1.54 (100) IL-21 0.56 1.868.46 23.85 9.20 6.39 8.43 3.20 (200)

In addition, additional endpoints were measured between groups. Thefollowing endpoints where compared: Body weight, spleen weight, vascularleakage in lung and liver, and serum cytokines. No significantdifference in body weights was observed between groups. As discussedabove, animals treated with both doses of IL-2, Group II and III, hadsignificantly heavier spleen weights as compared to IL-21 and PBScontrol treated animals (p<0.0001). Animals treated with both doses ofIL-21 treated animals, Group IV and V, had significantly heavier spleenweights as compared to PBS control animals (p<0.007 Group IV andp<0.0001 Group V).

Vascular leakage was also measured in both lung and liver. In lung, bothgroups of IL-2 treated animals, Group II and III, had a significantincrease in vascular leakage (p<0.0001) as compared to PBS controlanimals. Only Group III, the high dose of IL-2 had a significantincrease in vascular leakage as compared to both low dose and high doseIL-21 (p<0.0001 and p<0.0065) respectively. Only the highest dose ofIL-21, Group V, had a significant increase in vascular leakage ascompared to PBS treated animals (p<0.0001). However, the amount ofvascular leak was significantly lower than all of the IL-2 treatedanimals. In liver, both the low and high dose of IL-2 treated animalshad a significant increase in vascular leakage (p<0.0016 and p<0.0001respectively) as compared to PBS treated animals. Animals treated withthe high dose of IL-2 had a significant increase in vascular leakage ascompared to both low and high dose IL-21 treated animals (p<0.0002 andp<0.001 respectively). Only the low dose IL-21 treated animals had asignificant increase in vascular leakage as compared to PBS treatedanimals (p<0.0397).

Example 18 Flow Cytometric Analysis IL-21 Receptor Expression

The expression of IL-21 receptors on neoplastic B cells derived fromnon-Hodgkin's lymphoma (HL) specimens was assessed. Multiple MAbs wereused to identify neoplastic B cells and to co-localize IL-21 receptors(WIPO Publication No.s WO 0/17235 and WO 01/77171). Theimmunofluorescent staining by anti-IL-21R MAb or by biotin-IL-21 wasrecorded as mean peak fluorescence. The qualitative scores were assessedbased on the shift in mean peak fluorescence relative to an isotypematched control MAb.

Using either anti-IL-21 receptor MAb or biotin-IL-21 (U.S. Pat. No.6,307,024) we consistently detected IL-21 receptor on follicularlymphoma (FL) specimens derived from lymph node. However, nearly allspecimens derived from chronic lymphocytic leukemia (CLL) patients didnot show significant staining for IL-21 receptors, or staining at verylow intensity relative to negative control MAbs. The staining byanti-IL-21 receptor MAbs and biotin-IL-21 correlated well and detectedmoderate staining of follicular lymphoma. These data suggested thatIL-21 receptors represent a therapeutic target for follicular lymphoma.

Example 19 Activity of Mouse IL-2 1-Treated Alloreactive Murine CTL(Cytotoxicity Assays)

A. CTL Assay

CTL (cytotoxic T lymphocyte)-mediated target cytolysis was examined by astandard ⁵¹Cr-release assay. Alloreactive (H-2^(b) anti-H-2^(d)) CTLwere generated in a mixed lymphocyte culture with C57B1/6 splenocytes(H-2^(b)) with 3000 rad-irradiated Balb/c splenocytes (H-2^(d)). After 7days, the CTL were re-stimulated with irradiated Balb/c splenocytes (andno additional cytokines). After an additional 7 days, CTL werere-stimulated for 5 days in the presence of supernatants collected fromconA-activated rat splenocytes (a crude source of cytokines known tosupport CTL growth), 10 ng/ml recombinant mouse IL-2 (R&D Systems, Inc,Minneapolis, Minn.), recombinant human IL-15 (R&D Systems), IL-21, or acombination of IL-15 and IL-21 (5 ng/ml each). After 5 days, the CTLwere assayed for their capacity to lyse ⁵¹Cr-labeled target cells:H-2^(d) P815 mastocytoma cells (ATCC No.TIB-64) and the H-2^(b) thymomaEL4 (ATCC No.TIB-39) as a negative control.

We grew P815 and EL4 cells in RP10 medium (standard RPMI 1640(Gibco/BRL, Grand Island, N.Y.) supplemented with 10% FBS (Hyclone) aswell as 4 mM glutamine (Gibco/BRL), 100 I.U./ml penicillin+100 MCG/mlstreptomycin (Gibco/BRL), 50 μM β-mercaptoethanol (Gibco/BRL) and 10mMHEPES buffer (Gibco/BRL). On the day of assay, 1-2×10⁶ target cells wereharvested and resuspended at 2.5-5×10⁶ cells/ml in RP10 medium. We added50-100 μL of 5 mCi/ml ⁵¹Cr-sodium chromate (NEN, Boston, Mass.) directlyto the cells and incubated them for 1 hour at 37° C., then washed themtwice with 12 ml of PBS and resuspended them in 2 ml of RP10 medium.After counting the cells on a hemacytometer, the target cells werediluted to 0.5-1×10⁵ cells/ml and 100 μl (0.5-1×10⁴ cells) were mixedwith effector cells at various effector:target ratios. After a 4-hourco-incubation of effector cells and the labeled target cells at 37° C.,half of the supernatant from each well was collected and counted in agamma counter for 1 min/sample. The percentage of specific ⁵¹Cr releasewas calculated from the formula 100×(X−Y)/(Z−Y), where X is ⁵¹Cr releasein the presence of effector cells, Y is the spontaneous release in theabsence of effectors, and Z is the total ⁵¹Cr release from target cellsincubated with 0.5% Triton X-100. Data were plotted as the % specificlysis versus the effector-to-target ratio in each well.

CTL re-stimulated in the presence of rmIL-2 exhibited the highest lyticactivity on the P815 target cells, achieving >70% specific lysis at aneffector-to-target ratio of 33:1. The next most active CTL were thosere-stimulated in the presence of IL-21+rhIL-15 (62% specific lysis),followed by CTL cultured with rhIL-15 (˜50% lysis), CTL cultured withIL-21 alone (30% lysis), and CTL re-stimulated with rat conA supernatant(˜10% lysis). None of the CTL lysed the H-2^(b) EL4 cells (all CTL lysedfewer than 2% of the EL4 targets, even at the highest effector-to-targetratio of 33:1). This pattern of cytokine enhancement of cytolysis(IL-2>IL-21+IL-15>IL-15>IL-21>conA SN) held true in 2 replicateexperiments. These data demonstrate that IL-21, particularly incombination with IL-15, can enhance CTL effector function.

Example 20

Delayed Type Hypersensitivity in IL-21 Knockout (KO) Mice

IL-21 is a cytokine that is produced by T cells and has been shown toplay a role in T cell proliferation and function. Delayed TypeHypersensitivity (DTH) is a measure of helper CD4 T cell responses tospecific antigen. In this, mice are immunized with a specific protein(E.g., chicken ovalbumin, OVA) and then later challenged with the sameantigen in the ear. Increase in ear thickness after the challenge is ameasure of specific immune response to the antigen, mediated mainly byCD4 T cells. To understand the in vivo function of IL-21, mice deficientin IL-21 protein were engineered (IL-21 KO mice). If IL-21 is importantfor T cell responses, IL-21 KO mice should be expected to have a defectin T cell responses. One method to test this is to induce a DTH responsein IL-21 KO mice. IL-21 KO mice and control littermates were immunizedwith OVA mixed with an adjuvant CFA (Complete Freund's Adjuvant). Groupsof mice were then rechallenged in the ear with either PBS (control) orOVA. Control mice developed good DTH when injected with OVA as shown byincrease in the ear thickness at 24 hours post challenge. In contrast,IL-21 KO mice had a lesser degree of ear thickness compared to controls.This difference was statistically significant (p=0.0164). However, at 48hour post challenge, there was no difference in the response of wildtype or IL-21 KO mice. As expected, both control and IL-21 KO mice didnot respond to PBS (no change in ear thickness).

IL-21 KO mice (n=8) and control wild type littermates (n=8) wereimmunized in the back with 100 μg chicken ovalbumin (OVA) emulsified inCFA in a total volume of 200 μl. Seven days after the immunization, halfthe mice in each group (n=4/gp) were injected with 10 μl PBS in the earand the other half injected with 10 μg OVA in PBS in a volume of 10 μl.Ear thickness of all mice was measured before injecting mice in the ear(0 measurement). Ear thickness was measured 24 hours and 48 hours afterchallenge. The difference in ear thickness between the 0 measurement andthe 24 hour or 48 hour measurement was calculated.

At 24 hour post challenge, control mice or IL-21 KO mice rechallengedwith PBS showed minimal or no change in ear thickness. In response tothe OVA rechallenge, control mice ears showed significant inflammation(7.3±1.1×10⁻³ in). In contrast,, IL-21 KO mice showed a decrease in earthickness compared to controls (5±0.42×10⁻³ in). This difference wasstatistically significant (p=0.0164). This suggests that IL-21 does playan important role in CD4 T cell responses. However, at 48 hour postchallenge, responses in IL-21 KO mice were no different from controlssuggesting that IL-21 does not influence the response at this stage.Further experiments are underway to assess the role of IL-21 in DTHresponses and in T cell responses.

These results suggest that IL-21 plays an important role in CD4 T cellresponses. CD4 T cell responses contribute significantly to immunity,both in a positive manner to boost immunity towards microbes and tumorsand in a negative manner in cases of autoimmunity and inflammation. Useof IL-21 may be considered to boost CD4 T cell responses based on theabove results.

Example 21

IL-21 Modifies the Response of OT-1 T Cells to OVA Peptide as Presentedby Murine Dendritic Cells.

A. Isolation and Labeling of OT-1 T Cells

Mice bearing a transgenic T cell receptor specific for OVA257-264 inH-2Kb are available (OT-I transgenics, Jackson Laboratories). Cells fromlymph nodes from these animals were adherence depleted and the CD8 Tcells (OT-I T cells) were enriched by negative selection using CD8Cellect columns (Cedarlane Laboratories, Homby, Ontario, Canada). Purityof CD8 T cells was assessed by flow cytometry and was typically 90-95%with <1% CD4 T cells.

OT-I T cells were labeled with carboxyfluorescein diacetate succinimidylester (CFSE; Molecular Probes, Eugene, Oreg.) by placing them in growthmedia comprising RPMI-1640 medium supplemented with 10% FCS (JRH, LenexaKans.; Hyclone, Logan Utah), 2 mM glutamine (Gibco BRL), 50 U/mlpenicillin (Gibco BRL), 50 μg/ml streptomycin (Gibco BRL, Grand Island,N.Y.) and 50 μM 2-mercaptoethanol (Sigma, St Louis, Mo.) containing 5 μMCFSE for 5 min at room temperature. The cells were then washed threetimes, each time by resuspending in PBS containing 5% FBS, centrifuging5 min at 300′ g, 20° C., and removing the supernatant. Cells wereresuspended in growth media prior to use.

B. Preparation of Murine Dendritic Cells

Bone marrow derived dendritic cells (DCs) from mouse bone marrow werecultured in growth media the presence of GM-CSF using well-known methods(e.g., Inaba, K. et al., J. Exp. Med. 176:1693-1702, 1992). After sixdays in culture they were stimulated with 1 ug/ml LPS (Sigma, St Louis,Mo.) overnight and then washed in growth media prior to use.

C. In Vitro Stimulation of T Cells

DCs prepared as above were pulsed with 10 nm OVA257-264 peptide (SEQ IDNO:17) for 2 hours. The pulsed DCs are washed in growth media to removeany unbound peptide and then cultured with purified OT-I T cellsprepared as described above in the presence of either media alone or 20ng/ml mouse rIL-2 (R&D Systems, Minneapolis, Minn.) or 50 ng/ml murineIL-21 (U.S. Pat. No. 6,307,024). After either 48 or 72 hours ofincubation the cells were harvested and analyzed by flow cytometry forlevels of CFSE fluorescence and Annexin V binding (Pharmingen, SanDiego, Calif.) per manufacturers instruction.

Results showed that when OT-I T cells are presented specific antigen onDC's they undergo 3-5 rounds of cell division by day 2 and 5-7 rounds ofcell division by day 3 as evidenced with CFSE labeling. In the presenceof IL-2 their proliferation is increased such that by day 2 they havegone 5-6 rounds and by day 3, 7-9 rounds. When the T cells are treatedwith IL-2 they undergoing apoptosis at day 3 as demonstrated by AnnexinV binding. In contrast to IL-2, IL-21 induces increased T cellproliferation and prevents Annexin V labeling up to day 3. IL-21continues to enhance proliferation and prevent apoptosis even in thepresence of added IL-2.

IL-21 both enhances proliferation and reduces apoptosis of the murineCTL cells. This activity implies a positive immunostimulatory role forIL-21 in clinical settings, such as cancer or viral disease, where CTL'scan play a role.

Example 22

Murine IL-21 Effect on EG0.7 Thvmoma Growth In Vivo:

IL-21 Modifies the Response of OT-I T Cells in the EG-7 Model of CTLMediated Anti-tumor Activity

Cytotoxic T lymphocytes (CTL) recognize infected and transformed cellsby virtue of the display of viral and tumor antigens on the cellsurface. Effective anti-tumor responses require the stimulation andexpansion of antigen specific CTL clones. This process requires theinteraction of several cell types in addition to CTL and usually resultsin the establishment of immunologic memory. The EG-7 tumor cell line istransfected with chicken ovalbumin and thereby expresses a wellcharacterized T cell antigen, an ova peptide (SEQ ID NO: 17) presentedin H-2K^(b). OT-I T cells (Example 21) kill EG7 tumor cells in vitro andin vivo. (Shrikant, P and Mescher, M,. J. Immunology 162:2858-2866,1999).

Mice (female, C57B16, 9 weeks old; Charles River Labs, Kingston, N.Y.)were divided into three groups. On day 0, EG.7 cells (ATCC No. CRL-2113)were harvested from culture and 1,000,000 cells were injectedintraperitoneal in all mice. Mice were then treated with the testarticle or associated vehicle by intraperitoneal injection of 0.1 ml ofthe indicated solution. Mice in the first group (n=6) were treated withvehicle (PBS pH 6.0), which was injected on day 0, 2, 4, and 6. Mice inthe second group (n=6) were treated with murine IL-21, which wasinjected at a dose of 10 μg on day 0, 2, 4, and 6. Mice in the thirdgroup (n=6) were treated with murine IL-21, which was injected at a doseof 75 μg on day 0, 2, 4, and 6. In both groups of mice treated withmurine IL-21, time of survival was significantly increased, compared tomice treated with vehicle. The group treated with 75 μg doses of IL-21had significantly greater survival than the group treated with 10 μgdoses, and 33% (2/6 mice) of this group survived longer than 70 days. Anadditional portion of this study tested the effect of the same dosagescarried out through day 12. The results were very similar to the shorterdosing schedule, with both doses having significantly increased survivalover vehicle treatment, and the highest dose gave the best response (50%survival past 70 days).

In some experiments 4,000,000 OT-I T cells were injected intraperitonealin the mice on the day prior to day 0. The mice were then treated withIL-21 or vehicle as above. At various times after treatment OT-I T cellswere recovered from the peritoneal cavity and counted. The presence ofthe OT-I T cells had no effect on the survival time of the vehicletreated mice. IL-21 treatment resulted in a ten-fold increase in thenumber of OT-I T cells that could be recovered from the peritonealcavity. In mice treated with IL-21 the presence of the OT-I T cellsenhanced survival time compared to the mice treated with IL-21 alone.

The increase in survival conferred by IL-21 treatment with or with outexogenously added tumor specific T cells shows that IL-21 is activatingendogenous effector cells of the immune system. The increased recoveryof OT-I T cells from the peritoneal cavity shows that IL-21 isincreasing the number of tumor specific T cells at the site of thetumor.

As predicted by the ability of IL-21 to enhance T cell survival in vitrothese results indicate that treatment with IL-21 has enhanced theability of the immune system to destroy tumor cells in vivo. Theseresults in vivo demonstrate a positive immunostimulatory role for IL-21in relevant clinical settings, such as in human cancer or viral disease,where CTL's can play a role in combating disease.

Example 23

IL-21 Reduces Tumor Load in the RMA-RAE1 Model of NK Mediated Anti-tumorActivity

Natural killer cells serve as a first line of defense against certainviral infections and tumors. Effective NK cell activity does not requireprior exposure to the target nor are they thought to maintainimmunologic memory of the target. Thus, NK cells “sense” if cells aretransformed, infected, or otherwise “stressed” by virtue of a range ofmolecules on the surface of the target cell. RAE-1 is a proteinexpressed on the surface of “stressed” cells which specifically engagesan activating a receptor on the surface of NK cells thereby leading tolysis of the “stressed” cell. Transfection of the RMA tumor cell linewith RAE-1 renders it sensitive to lysis by NK cells both in vitro andin vivo.

The RMA lymphoma cell line (provided by Dr. L. Lanier and Dr. Jay Ryan,UCSF, San Francisco, Calif.) was grown in RPMI-1640 medium supplementedwith 10% FCS (JRH, Lenexa Kans.; Hyclone, Logan Utah), 2 mM glutamine(Gibco BRL), 50 U/ml penicillin (Gibco BRL), 50 μg/ml streptomycin(Gibco BRL, Grand Island, N.Y.) and 50 μM 2-mercaptoethanol (Sigma, StLouis, Mo.). Stable transfectants of RMA-RAE-1delta or mock-transfectedRMA cells were established by electroporation: 30 μg ofRAE-Idelta-pCDEF3 plasmid (RAE-1delta transfectant), or pCDEF3 plasmid(mock-transfectant) was added to about 1×10⁷ cells in RPMI-1640 mediumin a 4-mm cuveffe (BioRad, Richmond, Calif.), respectively. The pCDEF3vector was kindly provided by Dr. Art Weiss (UCSF, San FranciscoCalif.). Electroporation was performed by using a BioRad gene pulser(250 V, 960 μF). 48 h after electroporation, RMA-RAE-ldelta andmock-transfected cells were cultured in complete RPMI-1640 mediumsupplemented with 1 mg/ml G418 (GIBCO BRL).

Groups of six or more animals per experiment were injectedintraperitoneally with cells which were mock-transfected or transfectedwith RMA-RAE-1delta. Preliminary experiments titrating the number oftumor cells injected indicated that 1×10⁴ RMA cells and 1×10⁵ RMA-RAE-Idelta cells resulted in tumor formation and subsequent morbidity in100% of animals. Intraperitoneal (IP) inoculation of mice withRMA-RAE1delta cells at numbers comparable to lethal doses (about 1×10⁴)of the parental RMA cells results in the tumors being completelyrejected without the involvement of T cells or the establishment ofimmunologic memory. IP inoculation of mice with a 10 fold excess ofRMA-RAE1 delta cells results in death of the mouse presumably by‘swamping’ the capacity of the NK cells to reject the tumor (Cerwenka etal., Proc. Nat. Acad. Sci. 98:11521-11526, 2001). For cytokine efficacyexperiments six IP injections of 10 ug of murine IL-21 or vehiclecontrol were administered to the mice every other day on days −4, −2, 0,2, 4 and 6. All mice were monitored daily for tumor ascites development,indicated by swelling of the abdomen, and were sacrificed when tumorburden became excessive to avoid pain and suffering. Animals wereregarded tumor free when surviving longer than 8 weeks. For there-challenge experiments, surviving animals were inoculated with 1×10⁴RMA-mock cells after 8 weeks.

The administration of small amounts of murine IL-21 resulted in enhancedsurvival of mice receiving the 10 fold excess number of RAE1 bearingtumor cells. Some IL-21 treated mice became completely tumor-free. IL-21treatment had no effect on the survival of mice given the parental RMAcell line showing that the effect was specifically mediated by NK cells.It appeared that RAE1, and thereby NK cells, were required for the IL-21effect. Moreover, mice that survived the lethal RMA-RAE1 tumor challengeby virtue of IL-21 treatment were able to reject a subsequent challengewith the parental RMA cell line. This showed that IL-21 had also inducedimmunological memory in these mice. This ability of IL-21 to enhance theactivity of NK cells shows that IL-21 can have therapeutic benefit inthe treatment of patients who have tumors or viral diseases.

Example 24

Immunohistochemistry of IL-21 in Various Human Tissues and Cell Lines

The purpose of this experiment was to determine if IL-21 could bedetected in select tissues by means of immunohistochemistry. Tissueswere processed by standard immunohistochemical methods using a Techmate500 (BioTek Solutions, Tucson, Ariz.). Briefly, deparaffinized sectionsof paraffin-embedded tissues were treated with 5% normal goat serum inPBS and a blocking agent (Zymed Laboratories, Inc., South San Francisco,Calif., Reagent A and B (ready to use)) to minimize nonspecificbackground staining. One of two primary anti-IL-21 antibodies wasapplied (E3149 (mouse>human IL-21-CHO, HH4.9.1C2.1A6.1C8, PAS) or E2865(mouse>human IL-21-CHO, HH4.3.1.2D1.1C12, PAS), both made in house)followed by a biotinylated goat anti-mouse antibody (VectorLaboratories, Burlingame, Calif.). A colored reaction product wasgenerated via a peroxidase-3′3′-diaminobenzidine reaction (ChemMateperoxidase/DAB staining kit including methyl green counter stain;CMS/Fisher, Houston, Tex.). The slides coverslipped and then examinedunder a light microscope (Nikon Eclipse E600, Nikon Corporation, Tokyo,Japan).

The following cells and tissues were tested: BHK cells transfected withhuman IL-21 (positive control), BHK-570 cells, wild type (negativecontrol), and human normal lung, human lung with chronic perivascularinflammation, human normal lymph node, human lymph node with B celllymphoma, human spleen with myelofibrosis, and human duodenum. Thesetissues were obtained on a contract basis either from CHTN (Nashville,Tenn.) or NDRI (Philadelphia, Pa.). Also tested were normal humantissues on a multi-tissue slide (Biomeda, Hayward, Calif.) and humanabnormal/tumor tissues on a multi-tissue slide (Biomeda, Hayward,Calif.).

The E3149 antibody produced positive staining only in the transfectedBHK cells. With the E2865 antibody, intense staining was observed in thepositive control cells as well as occasional mononuclear cells ofunknown identity in the epithelium of the small intestine in the normalmulti-tissue block. The location in the small intestine from which thissample was obtained is unknown. This positive cell type was rare (1 cellin 3 sections) in a separate section of human duodenum.

Moreover, a positive population of mononuclear cells was diffuselydistributed throughout the human spleen from a patient withmyelofibrosis. A similar staining pattern was not found in the spleensof the normal multi-human tissue block. Although there was some stainingin the spleens of the multi-tissue block, the staining was not clearlycell associated. In addition, a section of inflamed lung containedstained spindle-shaped cells and mononuclear cells in what looks likethe subpleural space (the size and quality of section make determinationof location difficult). Positive staining was observed in scatteredpituicytes in pituitaries in the multitissue block. Isotype antibodystained pituitaries were negative. Colloid staining was observed inthyroid sections of the multi-tissue block and in a section of thyroidadenocarcinoma in the multi-tumor block. The significance of this isunknown-the colloid in the isotype section occasionally was stained (butmuch less intensely than in the corresponding anti-IL-21 stainedtissues). Light staining was also seen in the thyroid follicularepithelium, but its intensity was near background levels.

Staining in the undifferentiated carcinoma in the multi-tumor block isassociated with a central area of necrotic debris mixed withinflammatory cells-the specificity of this staining is questionable.Likewise, staining in the pancreatic adenocarcinoma may be associatedwith necrotic debris or associated inflammatory cells.

Because of the location of staining in the above tissues it is possiblethat IL-21 plays a role in intestinal mucosal immunity (occasional cellsin gut epithelium), inflammation (associated with inflammatory cells inlung, undifferentiated carcinoma, pancreatic adenocarcinoma) andmyelofibrosis; fibrosis in the bone marrow resulting in extramedullaryhematopoiesis in the spleen-could this also involve proliferation of thelineage of cells that produces IL-21 with IL-21 attempting to regulatethe process. A caveat with these results is that we get differentstaining patterns with different antibodies. This may be due to therecognition of different epitopes by the two antibodies.

Example 25 IL-21 Promotes IL-2 Stimulated NK-cell Expansion in PBMNCCultures in the Presence of IL-4

IL-4 inhibits the expansion of NK-cells stimulated with IL-2. In twoexperiments human peripheral blood mononuclear cells (PBMNCs) wereseeded at 200,000 cells/well in alpha-MEM+10% autologous serum with 10ng/ml IL-2 (R&D Systems, Minneapolis, Minn.) with or without 0.5 ng/mlIL-4 (R&D Systems, Minneapolis, Minn.), and with or without 10 ng/mlIL-21 (U.S. Pat. No. 6,307,024) and grown for 8 days. The number ofviable cells per well was determined using standard methods and thecells analyzed by flow cytometry for expression of CD3, CD16, and CD56.NK-cells were defined as the CD56 positive CD3 negative population.

The cultures from the two donors cultured with IL-2 alone containedabout 151,000 and 326,000 NK-cells respectively on day 8. The culturesfrom the two donors cultured with IL-2 and IL-21 contained about 446,000and 588,000 NK-cells respectively. The cultures from the two donorscultured with IL-2 and IL-4 contained about 26,000 and 29,000 NK-cellson day 8. However, cultures from the two donors cultured with IL-2, IL-4and IL-21 contained about 229,000 and 361,000 NK-cells representing an8.8 and 12.5 fold increase in NK-cell yield over the culture with IL-2and IL-4 only.

These results demonstrate that IL-21 promotes NK-cell expansion, andthat IL-21 can largely overcome the inhibitory effects of IL-4 onNK-cell growth. In some diseases IL-4 expression can play a role in thepathology. For example mice bearing the B16F10 melanoma generate a largepopulation of IL-4 producing CD4+ T-cells which appear to limit the hostanti-tumor response. Furthermore, STAT 6 (required for IL-4 signaling)gene deficient mice exhibit an enhanced ability to reject tumors. Theability of IL-21 to antagonize the action of IL-4, and induce theexpression of IFN-γ (described herein), in addition to the in vivoanti-tumor activity and data described herein, suggest that IL-21 can beuseful in treating malignacies, infections or autoimmune disease wherethere is a Th2 response limiting the hosts ability to control thedisease.

Example 26

IL-21 Synerzizes With IL-2 to Promote the Growth of NK Cells FromPeripheral Blood.

Peripheral blood lymphocytes from a healthy human donor were prepared bya standard Ficoll centrifugation method. Lymphocytes were magneticallynegatively enriched as described herein using the human NK cell negativeenrichment system from Stem Cell Technologies. NK's were cultured at astarting concentration of about 75,000/ml in 2 ml/well alpha MEM with10% donor serum, 50 μM BME, 2 ng/ml flt3L, and 0, 0.5, 10, or 50 ng/mlof IL-2±0, 5, or 50 ng/ml IL-21. After 15 days of culture cells wereharvested, counted, and analyzed by flow cytometry for CD3, CD56, andCD161. All cells analyzed after 15 days of culture were CD3−/CD56+,which are defined as NK cells. At day 0, cells were analyzed by flowcytometry and found to be >98% CD3−/CD56+.

The “fold increase” in cell number is defined as the final cell numberdivided by the starting cell number. Any “fold increase” below 1 istherefore a decrease in cell number. In general, the results at 10 ng/mlof IL-2 were similar to the results obtained with 50 ng/ml of IL-2, andthe results at 5 ng/ml IL-21 were similar to those obtained with 50ng/ml IL-21. With no IL-2 present, the fold increase in total cells was0.064. When 5 ng/ml IL-21 was included in the culture, the foldincreases was 0.11. This indicates that IL-21 has very littleproliferative activity on NK's by itself. At a low concentration of 0.5ng/ml IL-2, we saw a fold increase of 0.25. When 5 ng/ml IL-21 wasincluded, we saw a fold increase of 2.9. At higher concentrations ofIL-2 the fold increases were overall higher, but the effect of IL-21 wasin general decreased, although still positive. At 10 ng/ml IL-2 we saw afold increase of 2.9. When 5 ng/ml IL-21 was included, we saw a foldincrease of 7.

IL-2 1's effect in these cultures was dependent on the presence of atleast low dose IL-2. Without IL-2, the effect of IL-21 was minimal. WhenIL-2 is present, especially at the lower, perhaps physiological,concentration, IL-21's effect is the most profound. The lack of effectof IL-21 alone, coupled with its ability to synergize with lowconcentrations of other cytokines may allow it to act therapeutically atsites of infection or malignancy without causing systemic toxicity.

Example 27

IL-21 Stimulates Outgrowth of NK and NKT From Peripheral BloodLymphocyte Cultures That Contain IL-2 or IL-15

Peripheral blood lymphocytes from 3 healthy human donors were preparedby the standard Ficoll centrifugation method. Lymphocytes were thencultured at a starting concentration of 200,000/ml in alpha MEM with 10%donor serum, 50 μM BME (Sigma), 2 ng/ml flt3L (R&D Systems), and 0, 0.5,10, or 50 ng/ml of IL-2 (R&D Systems) or IL-15 (R&D Systems)±0, 5, or 50ng/ml IL-21 (U.S. Pat. No. 6,307,024). After 12 days of culture cellswere harvested, counted, and analyzed by flow cytometry for CD3, CD56,and CD8. NK cells were defined as CD56+/CD3− and NKT cells were definedas CD56+/CD3+.

The “fold increase” in cell number (defined as final cellnumber/starting cell number) was widely variable between the threedonors, but the trends were fairly consistent. In general, the resultsat 10 ng/ml of IL-2 or IL-15 were similar to the results obtained with50 ng/ml of IL-2 or IL-15, and the results at 5 ng/ml IL-21 were similarto those obtained with 50 ng/ml IL-21. With no IL-2 or IL-15 present,the fold increase in total cells was 0.33, 0.23, and 0.19 among thethree donors. When 5 ng/ml IL-21 was included in the culture, the foldincreases were 0.47, 0.31, and 0.35. At a low concentration of 0.5 ng/mlIL-2, we saw total cell fold increases of 2.2, 1.1, and 1.0 among thethree donors. When 5 ng/ml IL-21 was included, we saw total cellsincrease by 5.5, 2.3, 3.1. We saw fold increases in NK numbers withoutIL-21 of 16, 4.2, and 3.5. When IL-21 was present (at 5 ng/ml) theseincreases were 24, 15, and 21 respectively. NKT's were also positivelyeffected under these conditions. NKT fold increases were 4.4, 5.7, and1.8 without IL-21, and 10, 9, and 15 with 5 ng/ml IL-21.

These results are mirrored with IL-15. At 0.5 ng/ml IL-15, a total cellfold increases of 0.98, 0.43, and 0.88 was seen among the three donors.When 5 ng/ml IL-21 was included, we saw total cells increase by 1.4,0.9, 1.7 fold. Fold increases of NK numbers of 8.0, 0.85, and 3.7 wereseen without IL-21. When 5 ng/ml IL-21 was present, these fold increaseswere 13, 5.5, and 1 1. NKT fold increases at 0.5 ng/ml IL-15 were 3.3,2.3, and 1.6 for the three donors, but they were 3.9, 5.2, and 4.7 when5 ng/ml IL-21 was included.

At higher concentrations of IL-2 the fold increases were overall higher,but the effect of IL-21 was in general decreased, although stillpositive. At 10 ng/ml IL-2 we saw total cell fold increases of 18, 2.5,and 2.8 among the three donors. When 5 ng/ml IL-21 was included, we sawtotal cells increase by 21, 3.6, 9.8 fold. We saw fold increases in NKnumbers of 114, 13, and 13 without IL-21. When IL-21 was also present(at 5 ng/ml) these increases were 100, 19, and 56 respectively. NKT'swere also positively effected under these conditions. NKT fold increaseswere 33, 15, and 12 without IL-21, and 52, 20, and 38 with 5 ng/mlIL-21.

At 10 ng/ml IL-15, we saw total cell fold increases of 18, 0.8, and 1.7among the three donors. When 5 ng/ml IL-21 was included, we saw totalcells increase by 23, 1.4, 6.9 fold. We saw fold increases of NK numbersof 128, 0.58, and 2.0 without IL-21. When 5 ng/ml IL-21 was present,these fold increases were 107, 1.1, and 9.4. NKT fold increases at 10ng/ml IL-15 were 60, 6.5, and 5.7 for the three donors, but they were66, 12, and 33 when 5 ng/ml IL-21 was included.

IL-21's effects in these cultures were dependent on the presence of atleast low dose IL-2 or IL-15. Without those cytokines, the effect ofIL-21 was minimal. When IL-2 or IL-15 is present, especially at thelower, perhaps physiological, concentrations, IL-21's effect is the mostprofound. The lack of effect of IL-21 alone, coupled with its ability tosynergize with low concentrations of other cytokines may allow it to acttherapeutically at sites of infection or malignancy without causingsystemic toxicity.

Example 28

IL-21 Inhibits the Production of IL-13 in NK-Cell Cultures.

IL-13 shares receptor subunits and many of the biological activities ofIL-4, but unlike IL-4, IL-13 is produced by NK-cells. Since NK-cellsalso produce IFN-γ, and these two cytokines have in large part opposingactivities, experiments were conducted to examine the effects of IL-21on IL-13 and IFN-γ expression in PBMNC and NK-cell cultures.

Negatively selected human peripheral blood NK-cells were seeded at about3.75×10e5 cells/ml and stimulated 2days with 10 ng/ml IL-2, IL-4 (R&DSystems) or IL-21 (U.S. Pat. No. 6,307,024) or without any cytokine inalpha-MEM+10% autologous serum. After two days in culture IL-2 was addedto all wells to 10 ng/ml, and the cell were cultured for an additional 3days, then the supernatants were collected and analyzed by ELISA forIL-13 and IFN-γ. NK-cells grown for two days without any cytokineproduced about 2130 pg/ml IFN-γ and 175 pg/ml IL-13. Cells stimulatedwith IL-21 produced abut 10,300 pg/ml IFN-γ and 90 pg/ml IL-13. Cellsstimulated with IL-2 produced 12,700 pg/ml IFN-γ and 1000 pg/ml IL-13.Cells stimulated with IL-4 produced no detectable IFN-γ nor IL-13.

Of note, cells stimulated for the first two days with IL-21 produced 5times more IFN-γ, but only one half the IL-13 of unstimulated cells.When compared to cell stimulated with IL-2 for the first two days ofculture cells stimulated with IL-21 produced 80% as much IFN-γ, but only9% as much IL-13. Thus IL-21 selectively promotes the expression ofIFN-γ and depresses IL-13 expression.

Example 29

IL-21 Synergizes With IL-2 to Promote IFN-γ Production in Mouse SplenicNK Cells

C57BL/6 mouse splenic NK cells were prepared by water lysing a cellsuspension from the spleens, then utilizing the Stem Cell Technologiesmurine NK negative enrichment magnetic cell sorting protocol. The cellsprepared using this method were 65% Pan NK positive based on flowanalysis using the DX5 Pan NK antibody from PharMingen.

The negatively enriched murine NK cells were cultured for 8 days at500,000 cells/ml in RPMI 1640 with 10% heat inactivated fetal bovineserum and 2 mM L-glutamine, 50 μM BME, and PSN antibiotic with 20 ng/mlmIL-2 (R&D Systems) or 10 ng/ml mIL-21 (U.S. Pat. No. 6,307,024) orboth. The cell supernatants were harvested and cells were counted at theend of the culture period. Cell supernatants were assayed for mIFN-γusing a commercially available ELISA kit from PharMingen.

Cell numbers at the end of the eight day period were about 1,300,000 forthe IL-2 containing culture, 220,000 for the IL-2/IL-21 culture, and10,000 for the IL-21 culture. The mIFN-γ levels were 2.2 ng/ml, 30ng/ml, and 0.28 ng/ml respectively. When expressed as pg/500,000 cells,the results are 238, 14,000, and 12,000. IL-21 enhances IFN-γ expressionin these cultures, which when combined with the cellsurvival/proliferation effects of IL-2, results in high levels of IFN-γbeing secreted into the medium. IFN-γ is an important initiator of theimmune response, and is considered a THI biased cytokine. This datasupports that IL-21 plays a role in the anti-cancer, antiviral activityof the immune system, and hence can be used as a therapeutic inanti-cancer, anti-viral and other applications.

Example 30

Effects IL-21-Saporin Toxin Conjugate on T Cells and Human T Cell Lines

The ability of the murine IL-21-saporin toxin conjugate to bind normalmurine T Cells was determined by FACS competition assays and compared tobinding on the same cells by the murine IL-21. It was shown that themurine IL-21-saporin toxin conjugate binds to these cells with the sameaffinity as IL-21 (Example 30A).

The presence of Human IL-21 Receptor (WIPO Publication No.s WO 0/17235and WO 01/77171) on the following T Cell lines was determined by FACSanalysis: human T Cell leukemia MOLT-13 (DSMZ No. ACC_(—)436), humancutaneous T Cell lymphoma HUT-78 (ATCC No. TIB_(—)161), human cutaneousT Cell lymphoma HUT-102 (ATCC No. TIB_(—)162); human ALCL line DEL (DSMZNo. ACC_(—)338), and human T/NK cell leukemia YT (DSMZ No. ACC_(—)434;Example 30B).

The effects of human IL-21-saporin toxin conjugate described herein weretested on both normal human T Cells (Example 30C) and the Human T celllines shown to express the Human IL-21 Receptor (i.e., MOLT-13, HUT-78,HUT-102, DEL and YT; example 30D). The results showed that normal HumanT cells and T cell lines treated with IL-21-saporin toxin conjugateproliferated much less or not at all as compared with cells leftuntreated or cells cultured with unconjugated IL-21 or with saporinalone.

The results indicate that IL-21-toxin conjugate (saporin or other) cancontrol some types of T-cell neoplasms with little or no effect also onnormal Human T cell proliferation in vitro at 30 μM or less. A proposedmechanism of the observed inhibition of cell line proliferation in vitrois as follows: the Human IL-21-toxin conjugate binds with high affinityto IL-21 receptor expressed on the surface of these cells. The HumanIL-21-toxin conjugate is then taken up by the cells and, in the casewith the saporin-toxin conjugate, the cells' ability to produce proteinand their proliferation is subsequently blocked. Thus, IL-21-saporinimmunotoxin conjugate, or other IL-21-toxin fusion could be usedtherapeutically in prevention and treatment T-cell leukemias andlymphomas, and other cancers wherein IL-21 receptors are expressed.

A. The Binding of Murine IL-21-saporin Toxin Conjugate on Normal MurineT Cells by Flow Cytometry Analysis.

Total murine splenocytes were isolated from normal 4-month old femaleC57/BL6 mice (Jackson Laboratories, Bar Harbor, Me.). Spleens wereharvested and gently mashed between frosted slides to create a cellsuspension. Red blood cells were removed by hypotonic lysis as follows:cells were pelleted and the supernatant removed by aspiration. Wedisrupted the pellet with gentle vortexing, then added 900 ul of sterilewater while shaking, followed quickly (less than 5 sec later) by 100 ulof 10× HBSS (Gibco/BRL; Rockville Md.). The cells were then resuspendedin 10 ml of 1× HBSS and debris was removed by passing the cells over anylon mesh-lined cell strainer (Falcon/BD; Franklin N.J.). TheseRBC-depleted spleen cells were then pelleted and resuspended in FACSstain buffer: HBSS (GibcoBRL; Rockville Md.) containing 3% Human serum,1% BSA, and 10 mM HEPES.

Aliquots containing about 1×10⁶ white blood cells from spleen werestained for 3-color flow cytometric analysis with anti-murine CD3-FITC,anti-murine B220-CyChrome mAbs (PharMingen, San Diego, Calif.) andbiotinylated murine IL-21 (2 ug/ml) followed by Streptavidin-PE (Caltag;Burlingame Calif.). Staining of the biotinylated murine IL-21 wascompeted by equivalent titered molar amounts (0.0175 nM to 3.5 nM) ofboth unbiotinylated IL-21 and IL-21-saporin conjugate. Cells wereanalyzed on a FACSScan using CellQuest software (Becton Dickinson,Mountain View, Calif.). The results demonstrated that the murineIL-21-saporin toxin conjugate binds to these cells with the sameaffinity as IL-21.

B. The Binding of Biotinylated Murine IL-21 on Human T Cell Lines byFlow Cytometry Analysis.

Aliquots containing 0.4×10cxp6 to 1×10exp6 MOLT-13 cells, HuT-78 cells,HuT-102 cells, DEL cells or YT cells were stained for 1-color flowcytometric analysis with titered biotinylated murine IL-21 (40 ng/ml to1000 ng/ml) followed by Streptavidin-PE (Caltag; Burlingame Calif.).Cells were analyzed on a FACSScan using CellQuest software (BectonDickinson, Mountain View, Calif.). The results demonstrated thatbiotinylated murine IL-21 binds to these cell lines with strongaffinity. The results also demonstrated that the murine IL-21 iscross-species reactive as it recognizes and binds the Human IL-21Receptor molecule on the surface of the Human T cell lines.

C. The Effect of Human IL-21-saporin Immunotoxin on Normal Human T-cellProliferation.

Whole blood was collected from a healthy human donor, aliquoted into 50ml tubes and passed over Ficoll density gradients. RBC-depleted cells atthe interface (PBMC) were collected and washed extensively with PBSfollowed by RPMI 1640 supplemented with 10% human ultraserum and 2mM Lglutamine. The PBMC were suspended to 111×10exp6/ml in MACS buffer (PBS,1% BSA, 0.8mg/L EDTA). Cells were combined with anti-human CD14microbeads, anti-human CD19 microbeads, and anti-human CD56 microbeads(Miltenyi Biotech; Auburn Calif.) as per manufacturer specifications.The mixture was incubated for 20 min. at 4° C. These cells labeled withCD14, CD19 and CD56 beads were washed with 5× volume MACS buffer, andthen resuspended in 1.5 ml MACS buffer.

A VS+ column (Miltenyi Biotech; Auburn Calif.) was prepared according tothe manufacturer's instructions. The VS+ column was then placed in aVarioMACS™ magnetic field (Miltenyi Biotech; Auburn Calif.). The columnwas equilibrated with 3 ml MACS buffer. The CD14/CD19/CD56 bead-coatedPBMC were then applied to the column. The CD14−CD19−CD56− PBMC fractioncontaining Human T cells were allowed to pass through the column andwere collected in a 15 ml tube. The column was washed with 10 ml (2×5ml) MACS buffer to wash out residual Human T Cells. The column withbound CD14+CD19+CD56+ cells was discarded. The Human T cells and washeluant were pooled together and counted.

A sample of the negatively-selected human T cells was removed forstaining to assess the fraction's purity. A cychrome-conjugated mouseanti-human CD3 antibody (PharMingen) was used for staining the selectedcells. The negatively-selected T cells were shown to be 67% CD3+.

The isolated primary T cells were cultured at 0.5×10⁶/ml with equivalenttitered molar amounts (0.03 pM to 30 pM) of both IL-21 and IL-21-saporinconjugate on a plate coated with titered (0 to 2 μg/ml) anti-human CD3(clone UCHT-1; Southern Biotech; Birmingham Ala.) as a co-activator forthe Human T cells. After 3 days growth, the cells were pulsed with3H-thymidine (5 μCi/ml; AmershamBiosciences, Piscataway N.J.) for 18hours. Cells were lysed and DNA was captured onto glass filter mats(Packard, Meriden Conn.) and counted for 3H-thymidine incorporation toassess proliferation of the cells.

The results showed that IL-21 at 0.3 pM and higher had a stimulatoryeffect in combination with 2 μg/ml coated anti-CD3. The IL-21-saporinconjugate had no such stimulatory effect. It also had no inhibitoryeffect as compared with the 2 μg/ml anti-CD3 coat stimulus alone. Insum, these data can be interpreted to mean that while a IL-21 stimuluswas present in the wells containing the IL-21-saporin conjugate, theexpected increase in a proliferation due to the IL-21 part of themolecule was ablated by the presence of the saporin portion of theconjugated molecule. However, the saporin portion of the molecule didnot ablate the stimulatory effect due to the anti-CD3 coated antibody.

D. The Effect of Human IL-21-saporin Immunotoxin Conjugate on Human TCell Lines.

Cells were seeded at 5,000 cells/ml to 50,000 cells/ml with equivalenttitered molar amounts (0.2 pM to 400 pM) of both IL-21 and IL-21-saporinconjugate. After 2 days growth the cells were, pulsed with 5 μCi/ml³H-thymidine (AmershamBiosciences, Piscataway N.J.) for 18 hours. Cellswere either lysed and DNA was captured onto glass filter mats (Packard,Meriden Conn.) and counted for ³H-thymidine incorporation to assessproliferation of the cells.

IL-21-saporin treated MOLT-13 cells had incorporated ³H-thymidine toonly 70% of the ³H-thymidine incorporation by the IL-21 treated MOLT-13cells. IL-21-saporin treated HuT-78 cells grew to only 33% the densityof the untreated HuT-78 cells. IL-21-saporin treated HuT-102 cells grewto only 20% the density of the untreated Hut-102 cells. IL-21-saporintreated DEL cells grew to only 25% the density of the untreated DELcells. IL-21-saporin treated YT cells grew to only 33% the density ofthe untreated YT cells. The results indicate that IL-21-toxin conjugate(saporin or other) can be effective in controlling some types of T-cellneoplasms. Moreover, IL-21-saporin immunotoxin conjugate, or otherIL-21-toxin fusion could be used therapeutically in prevention andtreatment T-cell leukemias and lymphomas, and other cancers whereinIL-21 receptors are expressed.

Example 31

In Vivo Effects of IL-21 on B-cell Lymphomas

Human B-lymphoma cell lines are maintained in vitro by passage in growthmedium. The cells are washed thoroughly in PBS to remove culturecomponents.

SCID Mice are injected with (typically) one million human lymphoma cellsvia the tail vein in a 100 microliter volume. (The optimal number ofcell injected is determined empirically in a pilot study to yield tumortake consistently with desired kinetics.) IL-21 treatment is begun thenext day by either subcutaneous. implantation of an ALZET® osmoticmini-pump (ALZET, Cupertino, Calif.) or by daily i.p injection of IL-21or vehicle. Mice are monitored for survival and significant morbidity.Mice that lose greater than 20% of their initial body weight aresacrificed, as well as mice that exhibit substantial morbidity such ashind limb paralysis. Depending on the lymphoma cell line employed, theuntreated mice typically die in 3 to 6 weeks. For B cell lymphomas thatsecrete IgG or IgM, the disease progression can also be monitored byweekly blood sampling and measuring serum human Immunoglobulin levels byELISA.

A. IL-21 Dose Response/IM-9 Model

Mice were injected with 1×106 IM-9 cells, and 28 day osmotic mini pumpsimplanted the following day. The pumps were loaded with the followingconcentrations of IL-21 to deliver: 0, 0.12, 1.2 or 12 micrograms perday with 8 mice per dose group. IL-21 exhibited a clear dose dependenteffect in protecting mice from the tumor cell line. The effects of IL-21were dose dependent. Surviving mice at the end of the experiment had nosigns of disease and no detectable human IgG in their serum.

B. IL-21 NK Depletion/IM-9 Model

Mice were depleted of NK-cells by administering 5 doses ofanti-asialo-GM-1 antibody every third day beginning 15 days prior toinjection of tumor cells or left undepleted as controls. Half of thedepleted and undepleted mice were treated with 12 μg/day IL-21 and theother half were treated with vehicle only. Depletion of NK-cells did notsignificantly diminish the activity of IL-21. These data demonstratedthat NK-cells are not necessary for the effect of IL-21 in the IM-9model in SCID mice.

C. Other Cell Lines Tested

The following additional cell lines were tested using the model shownfor IM-9 cells. IL-21 delivered at 12 μg/day by minipump is effectiveagainst CESS cells in SCID mice. IL-21 administered to mice with RAJIcell implanted tumors had no efficacy. IL-21 administered to mice withRAMOS cell implanted tumors had no efficacy. IL-21 administered to micewith HS SULTAN cell implanted tumors had significant efficacy, but didnot prevent disease in most mice, only slows its onset. IL-21 DoHH2 hadno efficacy.

These data demonstrate that the efficacy of IL-21 in SCID mouse lymphomamodels correlates with the ability to inhibit the growth of the lymphomacell lines in vivo. Furthermore, NK-cell depletion of SCID mice for bothT-cells and B-cells does not diminish the effectiveness of IL-21 in theIM-9 model. It is likely that the efficacy of IL-21 in SCID mouselymphoma models is dependent on it direct effects on the tumor cellsbecause no efficacy was seen in three of three cell lines tested in themodel that were not inhibited by IL-21 in vitro, and NK depletion had noeffect on the efficacy of IL-21 in the IM-9 model. In a patient with anintact immune system, IL-21 dependent effector cell mediated antitumoreffects are predicted from experiments with immunocompetent mice insyngeneic tumor models. The demonstration of direct antitumor effects inSCID mice suggests that IL-21 therapy could have combined direct andeffector mediated anti-tumor effects in selected B-cell malignancies inhumans.

Example 32

The Effects of IL-21 in a Mouse Syngeneic Ovarian Carcinoma Model

The effect of IL-21 is tested for efficacy in ovarian carcinoma using amouse syngeneic model as described in Zhang et al., Am. J. of Pathol.161:2295-2309, 2002. Briefly, using retroviral transfection andfluorescence-activated cell sorting a C57BL6 murine ID8 ovariancarcinoma cell line is generated that stably overexpresses the murineVEGF164 isoform and the enhanced green fluorescence protein (GFP). Theretroviral construct containing VEGF164 and GFP cDNAs was transfectedinto BOSC23 cells. The cells are analyzed by FACS cell sorting and GFPhigh positive cells are identified.

The ID8 VEGF164/GFP transfected cells are cultured to subconfluence andprepared in a single-cell suspension in phosphate buffer saline (PBS)and cold MATRIGEL (BD Biosciences, Bedford, Mass.). Six to eight weekold femal C57BL6 mice are injected subcutaneously in the flank at 5×10⁶cells or untransfected control cells. Alternatively, the mice can beinjected intraperitoneally at 7×10⁶ cells or control cells. Animals areeither followed for survival or sacrificed eight weeks after inoculationand evaluated for tumor growth. Mice are treated with recombinant.murine IL-21 beginning 3-14 days following tumor implantation, or whentumor engraftment and growth rate is established. Treatment levels of0.5-5 mg/kg will be administered on a daily basis for 5-14 days, and maybe continued thereafter if no evidence of neutralizing antibodyformation is seen.

Example 33

The Effects of IL-21 in a Mouse RENCA Model

The efficacy of IL-21 in a renal cell carcinoma model is evaluated usingBALB/c mice that have been injected with RENCA cells, a mouse renaladenocarcinoma of spontaneous origin, essentially as described inWigginton et al., J. Nat. Cancer Instit. 88:38-43, 1996.

Briefly, BALB/c mice between eight and ten weeks are injected with RENCAcells R 1×10⁵ cells into the kidney capsule of the mice. Twelve daysafter tumor cell implantation, the mice are nepharectomized to removeprimary tumors. The mice are allowed to recover from surgery, prior toadministration of IL-21. Mice are treated with recombinant. murine IL-21beginning 3-14 days following tumor implantation, or when tumorengraftment and growth rate is established. Treatment levels of 0.5-5mg/kg will be administered on a daily basis for 5-14 days, and may becontinued thereafter if no evidence of neutralizing antibody formationis seen. Alternatively, RENCA cells may be introduced by subcutaneous(5×10e5 cells) or intravenous (1×10⁵ cells) injection.

The mice are evaluated for tumor response as compared to untreated mice.Survival is compared using a Kaplan-Meier method, as well as tumorvolume being evaluated.

Example 34

The Effects of IL-21 in a Mouse Colorectal Tumor Model

The effects of IL-21 in a colorectal mouse model are tested as describedin Yao et al., Cancer Res. 63:586-592, 2003. In this model, MC-26 mousecolon tumor cells are implanted into the splenic subcapsul of BALB/cmice. After 14 days, the treated mice are administered IL-21. Mice aretreated with recombinant. murine IL-21 beginning 3-14 days followingtumor implantation, or when tumor engraftment and growth rate isestablished. Treatment levels of 0.5-5 mg/kg will be administered on adaily basis for 5-14 days, and may be continued thereafter if noevidence of neutralizing antibody formation is seen.

The efficacy of IL-21 in prolonging survival or promoting a tumorresponse is evaluated using standard techniques described herein.

Example 35

The Effect of IL-21 in a Mouse Pancreatic Cancer Model

The efficacy of IL-21 in a mouse pancreatic cancer model is evaluatedusing the protocol developed by Mukherjee et al., J. Immunol.165:3451-3460, 2000. Briefly, MUC1 transgenic (MUC1.Tg) mice are bredwith oncogene-expressing mice that spontaneously develop tumors of thepancreas (ET mice) designated as MET. MUC1.Tg mice. ET mice express thefirst 127 aa of SV40 large T Ag under the control of the rat elastasepromoter. Fifty percent of the animals develop life-threateningpancreatic tumors by about 21 wk of age. Cells are routinely tested byflow cytometry for the presence of MUC 1. All mice are on the C57BL/6background. Animals are sacrificed and characterized at 3-wk intervalsfrom 3 to 24 wk. Mice are carefully observed for signs of ill-health,including lethargy, abdominal distention, failure to eat or drink,marked weight loss, pale feces, and hunched posture.

The entire pancreas is dissected free of fat and lymph nodes, weighed,and spread on bibulus paper for photography. Nodules are counted, andthe pancreas is fixed in methac am, processed for microscopy byconventional methods, step sectioned at 5 μm (about 10 sections permouse pancreas), stained with hematoxylin and eosin, and examined bylight microscopy. Tumors are obtained from MET mice at various timepoints during tumor progression, fixed in methacarn (60% methanol, 30%chloroform, 10% glacial acetic acid), embedded in paraffin, andsectioned for immunohistochemical analysis. MUC1 antibodies used areCT1, a rabbit polyclonal Ab that recognizes mouse and human cytoplasmictail region of MUC1, HMFG-2, BC2, and SM-3, which have epitopes in theTR domain of MUC1.

Determination of CTL activity is performed using a standard ⁵¹Cr releasemethod after a 6-day in vitro peptide stimulation without additionaladded cytokines. Splenocytes from individual MET mice are harvested bypassing through a nylon mesh followedby lysis of RBC.

Single cells from spleens of MET mice are analyzed by two-colorimmunofluorescence for alterations in lymphocyte subpopulations: CD3,CD4, CD8, Fas, FasL, CD11c, and MHC class I and II. Intracellularcytokine levels were determined after cells are stimulated with MUC1peptide (10 μg/ml for 6 days) and treated with brefeldin-A (also calledGolgi-Stop; PharMingen) as directed by the manufacturer's recommendation(4 μl/1.2×10⁷ cells/6 ml for 3 h at 37° C. before staining). Cells arepermeabilized using the PharMingen permeabilization kit and stained forintracellular IFN-γ, IL-2, IL-4, and IL-5 as described by PharMingen.All fluorescently labeled Abs were purchased from PharMingen. Flowcytometric analysis was done on Becton Dickinson FACscan using theCellQuest program (Becton Dickinson, Mountain View, Calif.).

Mice are treated with recombinant. murine IL-21 beginning 3-14 daysfollowing tumor implantation, or when tumor engraftment and growth rateis established. Treatment levels of 0.5-5 mg/kg will be administered ona daily basis for 5-14 days, and may be continued thereafter if noevidence of neutralizing antibody formation is seen.

Example 36

The Effects of IL-21 in a Murine Breast Cancer Model

The efficacy of IL-21 in a murine model for breast cancer is made usinga syngeneic model as described in Colombo et al., Cancer Research62:941-946, 2002. Briefly, TS/A cells which are a spontaneous mammarycarcinoma for BALB/C mice. The cells are cultured for approximately oneweek to select for clones. The selected TS/A cells are grown and used tochallenge CD-1 nu/nu BR mice (Charles River Laboratories) by injected2×10² TS/A cells subcutaneously into the flank of the mouse.

Mice are treated with recombinant. murine IL-21 beginning 3-14 daysfollowing tumor implantation, or when tumor engraftment and growth rateis established. Treatment levels of 0.5-5 mg/kg will be administered ona daily basis for 5-14 days, and may be continued thereafter if noevidence of neutralizing antibody formation is seen. The tumors areexcised after sacrificing the animals and analyzed for volume and usinghistochemistry and immunohistochemistry.

Example 37

The Effects of IL-21 in a Murine Prostate Cancer Model

The effects of IL-21 on tumor response are evaluated in murine prostatecancer model, using a model similar to that described in Kwon et al.,PNAS 96:15074-15079, 1999. In this model, there is a metastaticoutgrowth of transgenic adenocarcinoma of mouse prostate (TRAMP) derivedprostate cancer cell line TRAMP-C2, which are implanted in C57BL/6 mice.Metastatic relapse is reliable, occurring primarily in the draininglymph nodes in close proximity to the primary tumor.

Briefly, the C2 cell line used is an early passage line derived from theTRAMP mouse that spontaneously develops autochthonous tumorsattributable to prostate-restricted SV40 antigen expression. The cellsare cultured and injected subcutaneously into the C57BL/6 mice at2.5-5×10⁶ cells/0.1 ml media. Mice are treated with recombinant. murineIL-21 beginning 3-14 days following tumor implantation, or when tumorengraftment and growth rate is established. Treatment levels of 0.5-5mg/kg will be administered on a daily basis for 5-14 days, and may becontinued thereafter if no evidence of neutralizing antibody formationis seen. The tumors are excised after sacrificing the animals andanalyzed for volume and using histochemistry and immunohistochemistry.

Example 38

The Effects of IL-21 and Chemotheralpeutics on Growth of Human B-CellLines In Vitro

The effects of IL-21 and zeocin alone and in combination were tested onthe growth of IM-9 and HS Sultan human B-cell lines in vitro toascertain if the growth inhibitory/cytotoxic effects of IL-21 and achemo-therapeutic agent will be additive or synergistic on IL-21sensitive cells lines. ZEOCIN (Invitrogen, Carlsbad, Calif.) is anantibiotic with a mechanism of action similar to the relatedchemo-therapeutic bleomycin was used.

IM-9 and HS Sultan cell lines were plated at 50,000 cells/ml in RPMI1640medium suppplemented with 2 mM L-glutamine and 10% heat inactivated FBSwith and without IL-21 (at 20 ng/ml) and or ZEOCIN (at 15.6 μg/ml forIM-9s and 31 μg/ml for HS Sultans) for two days then the cells wereharvested and washed once to remove the zeocin and replated with orwithout IL-21 in a serial cell dilution series, with 6 wells perdilution, in 96 well round bottom plates to determine their relativegrowth ability. The plates were scored in 6 days using Alamar blue, as ameasure of viable cells per well, and reflecting their survival andoutgrowth from the prior treatment. Cells populations treated withZEOCIN had less than one tenth the growth capacity of untreated cells,and the combination of IL-21 with zeocin further reduced the growthcapacity by approximately an order of magnitude. These data suggest thatIL-21 could be successfully combined with chemotherapy to augmentresponse rates in the treatment of lymphoma.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method of tumor vaccination comprising administering to a subjectin need thereof an IL-21 polypeptide comprising residues 32 (Gln) to 162(Ser) of SEQ ID NO:2 in combination with a tumor antigen expressed frominjected DNA from breast cancer.
 2. The method according to claim 1,wherein the polypeptide has been shown to not cause proliferation ofisolated cancer cells prior to administration to the subject.
 3. Themethod of claim 1, wherein there is a tumor response.
 4. The method ofclaim 3, wherein a tumor response is measured as complete response,partial response or reduction in time to progression.